Flowjo

Data Analysis Software for Flow Cytometry

Version X User Documentation

Windows/Mac user documentation

8 Color PBMC Tutorial

Revision Date: 14 May 2013

version 10.0.6
Flowjo was written by Adam Treister and Mario Roederer beginning in 1996, based on concepts developed at the
Herzenberg Laboratory at Stanford. We are indebted to our active and enthusiastic users worldwide for their ideas,
discussions and tireless testing of new versions.

Flowjo, its tutorials, documentation and website are copyright © Tree Star, Inc. 1997-2013. All rights reserved.

• 8 Color PBMC •
• © MMXII •

Revision Date: 14 May, 2013

Version 10.0.6

i
Introduction
FlowJo is a software application with an integrated environment for viewing and analyzing flow cytometric data.
The environment is presented as the Workspace which contains a list of all of loaded samples (experimental data),
statistics, gates, other analyses, as well as tabular and graphical layouts. The Workspace is saved as a FlowJo document
on your hard disk; when you reopen the document, you will see your analysis as it was when it was last saved.

This tutorial is designed to cover a majority of the features pf the program. Reading through it, you will learn how
to operate FlowJo. Run the program as you perform the steps in the tutorial, so that you can get the best feel for
how the program works. As you watch FlowJo perform various operations such as creating new graphs, statistics,
tables, or graphical layouts, you will see how fast and easy FlowJo is to use.

FlowJo is capable of much more that simply can’t be covered in a tutorial like this (for example, there are analysis
platforms for DNA/Cell Cycle analysis, Kinetics, Proliferation, etc. and tools to export raw gated data for analysis
in other programs, etc.). You can learn more about FlowJo through our online help documents.

Pressing the question mark button in any window in FlowJo will launch a web browser and and access a help
pages relevant to that window. From this website, you can navigate the help pages to learn more about FlowJo. In
addition, FlowJo.com contains a page for FlowJo FAQs, tutorial videos, and the Daily Dongle, a blog discussing all
things FlowJo.

As a note, we are pleased to be able to frequently update FlowJo to provide new features and analysis capabilities.
Therefore, it is possible that the graphics shown in this tutorial may not exactly match the windows that you see
when you run the most recent version of FlowJo. You can download the most recent version of FlowJo from:

http://www.FlowJo.com/home/windows.html

This tutorial is designed to walk you through the data analysis process in FlowJo from adding data to producing
tables of statistics and publication-quality graphics. The data is from 2 8-color immunophenotyping experiments
of human peripheral blood mononuclear cells (PBMC). Keep in mind that the workflow described in this tutorial
will help you in the analyses of any kind of flow cytometric data.

The tutorial is divided into 11 lessons so you may perform it stepwise. The tutorial is written for the user to perform
all operations from start to finish, but we have included completed workspaces so that you can jump to any stage of
the tutorial. If you would like to perform the tutorial starting with a lesson other than 1, just open the workspace
from the preceding lesson and you will have all of the work completed through that lesson.

ii
 Table of Contents

About FlowJo..........................................................................................i
Introduction............................................................................................ii
Table of Contents...................................................................................iii

Getting Started.......................................................................................1
The Experimental Data.................................................................3
FlowJo Workflow...........................................................................3
Lesson 1: Workspaces and Basic Data Display.........................5
Opening a New Workspace..........................................................5
Adding Data...................................................................................6
Displaying and Editing Metadata................................................7
Adding a Keyword.........................................................................9
Displaying a Graph........................................................................10
Graph Types...................................................................................10
Three-Dimensional Polychromatic Plots ...................................11
Adjusting the Scale........................................................................12
Lesson 2: Gating and Statistics........................................................14
Creating a Polygon Gate...............................................................14
Editing a Gate.................................................................................15
Creating a Gate on a Histogram..................................................16
Adding Statistics............................................................................17
Other Types of Gates.....................................................................18
Deleting Gates................................................................................19
Lesson 3: Copying Analyses to Other Samples .........................20
Setting up an Analysis...................................................................20
Copying a gate to Another File....................................................20
Copying Multiple Gates................................................................21
Deleting Analyses..........................................................................22

iii
Lesson 4: Groups....................................................................................23
Creating a New Group..................................................................23
Modifying an Existing Group .....................................................24
Deleting a Group...........................................................................24
Creating a Group Using Keywords..............................................24
Adding Analyses to All members of a Group............................25
Modifying Group Gates................................................................26
Lesson 5: Tables and Collating Data Output.............................28
Creating a New Table....................................................................28
Deleting a Column........................................................................29
Ordering the Samples in a Table..................................................29
Renaming the Columns in a Table..............................................29
Adding a Keyword or Formula to a Table..................................30
Batching to Create a Table............................................................31
Table Iteration................................................................................33
Additional Tools............................................................................33
Applying Conditional Formatting...............................................33
Dynamic Updating .......................................................................33
Lesson 6: Creating Simple Graphical Layouts...........................34
Creating a New Layout..................................................................34
Creating a Second Graph of a Population..................................35
Editing a Graphs Appearance......................................................35
Aligning Plots.................................................................................35
Dragging Statistics into the Layout Editor.................................36
Creating Formulas in the Layout Editor.....................................36
Drawing Tools................................................................................36
Overlays..........................................................................................37
Ancestry and Backgating..............................................................38
Multigraph Overlays.....................................................................39
Displaying Adjunct Histograms..................................................40
Exporting a Layout Page...............................................................40

iv
Lesson 6: Continued...
Printing out of the Layout Editor................................................41
Copying and Pasting Graphs from FlowJo.................................42
Lesson 7: Creating Batch Graphical Reports..............................43
Batch Plotting.................................................................................43
Other Batch Outputs.....................................................................44
Lesson 8: Generating Complex Batch Reports..........................46
Varying Iteration Options............................................................46
Batching Overlays with Multiple Samples..................................48
Batching While Maintaining a Control Sample........................49
Lesson 9: Creating Finished Reports.............................................50
Importing a Graphic into FlowJo................................................50
Adding Text....................................................................................50
Adding Plots...................................................................................51
Using the Grid Tool.......................................................................52
Adding a Table from the Table Editor to the Layout Editor....53
Lesson 10: Compensation..................................................................54
Preference settings for loading data............................................54
Data Compensated by the Acquisition Software.......................54
Editing an Existing Matrix...........................................................56
Creating a New Matrix..................................................................57
Lesson 11: Setting Preferences..........................................................60
What are preferences?...................................................................60

v
Getting Started
To get started using FlowJo, you will first need to install the software. The easiest way to do this is to download the
most current installer which you can find on our website at: http://www.FlowJo.com/download/index

Select your platform (Mac or PC) at the top of the page.

For a PC, download the installer.exe file and open it to

install the program. Choose to place a shortcut on the
desktop, and when the installer finishes, double click
on the FlowJo shortcut icon to launch the program.

For a Mac, download the installer.zip file and double

click it to extract the program. Once the .zip file is
extracted, double click the icon to launch FlowJo.

When you launch FlowJo for the first time, the

FlowJo license information window will pop up. You
must select and input the type of license you have and
agree to the license terms.

For the use of running our tutorials, you need only to

select “Continue under free demonstration license”
and click done.

For more information about licenses, visit:

www.flowjo.com/home/licenseoptions

If you have a dongle (a physical

thumb drive that has a license
key on it) plugged into your
computer, FlowJo will detect it
automatically and allow you to
activate the program.

For more about dongles and for dongle support, visit:

www.flowjo.com/support/index.php?content=dongle

If you have an enterprise group license, you can

configure the connection here as well.

If you have an individual license that you would like

to use, input your serial number in the Serial Number
field (shown at right).

Demonstration data and the accompanying

workspaces can be acquired from our website:
http://www.flowjo.com/home/tutorials

Following this link will bring you to a page where you can download our Basic Tutorial and Advanced 8 Color
Tutorial as .zip files with .FCS (flow cytometric standard) files and a PDF of the tutorial. Save these files to disk and
double-click on the compressed file to extract them on a Mac and right click to extract them on a PC.

1
2
The Experimental Data
The demonstration data set is comprised of three folders :
• A folder of compensation controls.
• A folder named Exp. 1 which contains two subfolders named Panel 1 and Panel 2 which each contain 9
samples stained with two different combinations of reagents (see table below).
• A folder named Exp. 2 which contains two subfolders named Panel 1 and Panel 2 which each contain 9
samples stained with two different combinations of reagents, similar to Experiment 1.

There are 2 experiments, 2 panels, 9 tubes for each panel, and 7 compensation control tubes for a total of 43 tubes
(2x2x9+7). The compensation controls are provided for the user who wants to learn more about the compensation
editor (described in Lesson 10). However, all the files were compensated at the time of acquisition using Becton
Dickinson’s Diva™ software. FlowJo reads the acquisition compensation matrix from the files and displays the
compensated data by default. The stain combinations are listed by panel in the table below.
Panel No. APC-Ax 700 APC-H7 APC-Ax 647 Viability Dye Fitc PE Pe-Cy5.5 Pe-Cy7
1 CD3 CD19 2H2 7 AAD CD32 CD86 CD209 CD14
2 CD4 CD8 2H2 7 AAD CD56 CD11c CD3 CD45RO
Table 1: Experimental data panels
FlowJo Workflow

When you open FlowJo, the interface that appears

is referred to as a Workspace. It serves as :
• the repository for your data
• a tool for organizing the data
• a means for displaying and managing
your gating strategy
• a statistical display
• a starting point for all analyses

Double-clicking on a file opens a Graph Window. This

interface is used to view and set gates on individual tubes.

Fig. 1 - The workspace

The Table Editor is accessed through either the Table menu or through a
shortcut button at the top of the workspace. But the Table Editor button
is used from the populations and statistics created in the workspace (producing
tabular outputs.)

Fig. 2 - Graph window

Fig. 3 - Table editor

3
The Layout Editor is accessed through either the Layout menu or through a shortcut button at the top of the
workspace. It is used to create publication quality graphics.

Fig. 4 - Layout editor

All of these functions are designed based on the idea that each data file is part of an experiment and has context.
For that reason, many of the operations you will perform in FlowJo can be “batched”, that is, applied to a set of
files that the user wishes to analyze, print, or tabulate in a common manner. Batching enables you to perform
an operation once and instruct the software to repeat the action for a specified set of files, sparing the user the
repetitive work. If you find yourself repeating an action frequently it’s likely that a batch operation exists that will
save you significant time.

•••

4
Lesson 1: Workspaces and Basic Data Display
In this lesson you will learn how to start the program, load and organize your data, view graphs and other basic
concepts of the workspace.

The workspace in FlowJo provides an integrated environment for the viewing and analysis of flow cytometric data.
The workspace contains a list of the samples that you have loaded into FlowJo, gates applied, statistics calculated,
and the tables and graphical layouts that you have designed. If you save your workspace, you will save all aspects.

The workspace does not contain the raw data. Instead, the workspace saves pointers to the loaded data, avoiding
the problem of creating multiple copies of data files, which would use two or three times more memory on your
computer. Because FlowJo operates in this manner, if you move the raw data files to another disk, FlowJo will
prompt you to find the data the next time it needs that information. Workspaces store much of the information
about samples, as well as all of the analyses (gates and statistics) that you have previously computed. The best
practice is to save your analyzed workspaces in the same folder, at the same level, as the raw .fcs data files.

Think of the workspace as an experimental notebook. In general, you will create a different workspace for each
experiment that you perform. Workspaces may contain multiple samples collected on various days – and can
provide an efficient way to organize your data analyses.

To begin analyzing data in FlowJo, you will need to create a New Workspace and add data files into it. Later, you
can simply double-click on this Workspace to continue your work.

Opening a New Workspace

Launch FlowJo by double-clicking on the

FlowJo shortcut icon from the desktop. TASK BAR
Once you have launched FlowJo, Click
on the FlowJo tab, you will be shown a
window similar to the graphic to the right.
RIBBONS
Note the Question Mark icon at the far right
end of the menu choices. This is the FlowJo
Help menu. When clicked, this button GROUPS
displays options for help in specific topics,
which launch FlowJo’s web browser online.

The workspace window is divided into

four parts. The top portion is a taskbar.
The taskbar has action buttons to let you
add components to the workspace, such as
samples, groups, tables, etc. As you move SAMPLES
the mouse over an action button, FlowJo
displays a description of that button’s
action in the tool tip.

L1 fig. 1 - Workspace

5
 The actions associated with each button are:

New Workspace : Creates a new document and window.

Add samples : One means of adding data to the workspace

Add statistic : Opens a menu to add statistics to a selected node

New group : Opens an interface for organizing data

Table Editor : Opens an interface for creating tables of statistics

Layout Editor : Opens an interface for creating graphical reports

The second part of the workspace is a ribbon bar. This bar has action buttons that can be customized, which will be
discussed later. As you move the mouse over each action button, FlowJo displays a description of that action. The
third part of the workspace is a list of the current groups. At the moment the only groups that you will see in your
workspace are the All Samples Group and a Compensation Group. These are the default groups that are always
present; All Samples includes all of the data files in the workspace. Groups will be discussed in depth later in this
tutorial, specifically creating new groups, editing existing groups, and using groups to facilitate batch processing.

The bottom part of the window displays individual data files. The files in a selected group are displayed here. At the
moment there is no data in the workspace, so the area will be blank.

Adding Data

Locate the 8-Color PBMC tutorial folder that you downloaded, and double-click to open the folder. You will see the
Compensation, Exp.1 and Exp. 2 folders. If you open the Exp. 1 folder you will see the Panel 1.1 and Panel 1.2 folders.
If you open either of these you will see the Flow Cytometric Standard (FCS) files. The same file type a cytometer
outputs. To load Experiment 1 into FlowJo, click on the “Exp.1” folder, hold down the mouse button, and then drag
the folder to the bottom portion of the FlowJo Workspace. Release the mouse button.

L1 fig. 5 - Workspace
L1 fig. 2,3,4 - Samples

6
Your workspace will now populate with two additional groups and 18 total data files. It will look like the Workspace in
figure below (fig. 6).

Note that FlowJo will display the files

contained in whichever group you select.
Here we have selected the All Samples
group, so the files in All Samples are
displayed in the lower portion of the
workspace. If you click on a different
group, only the samples in that group will
be displayed. The number of samples in
each group is shown to the right of the
group name.

You will note that FlowJo has preserved

the file organization of the folders in its
groups. A new group was created for each
subfolder and the files that were stored in
the subfolder were automatically added to
the group of the same name. Additionally,
all of the files were added to the All
Samples Group.

Also note that each file is in more than

one group – the All Samples Group, Exp. 1
Group, and one of the panel-based groups
depending on the file. You can place a file
L1 fig. 6 - Samples in as many groups as you like. To add a file
to another group, you can click on the “All Samples Group”, select a file, then drag and drop it into another group.
You can also remove a file from a group by clicking on the group, locating the unwanted file, selecting it, and
pressing “delete” on your keyboard. The file will not be removed from the Workspace, only that particular group.
To remove a file from the Workspace entirely you must delete it from the All Samples Group.

Alternatively, to add data into the workspace you can click on the “Add Samples” shortcut button
at the top of the workspace. This button opens a standard Open File dialog. At that point you must navigate to the
data location and choose “Open”.

Displaying and Editing Metadata

FCS files contain a text portion with experimental information, or metadata, such as the type of cytometer on
which the data was collected, date collected, etc. This information takes the form of keywords; a set of code words
or abbreviations that are associated
with specific information. For example,
the keyword $DATE lists the date that
the data in an FCS file was collected.

Display some of these keywords in our

FlowJo workspace by clicking on the
“Configure” tab located at the top of the
workspace, and choosing the first item
in the band: “Edit Columns” (fig. 7).
L1 fig. 7 - Configure
7
The window shown at right will appear (fig. 8).

The left column in the window is a list of

all the keywords contained in the FCS file.
To display any of these in the workspace,
click on the chosen keyword, then press
the “Add Column” button. To remove
a keyword, click the keyword name in
the right column and select “Remove
Column” from the dialogue box. Note
that there is a “Default” button toward
the bottom of this dialogue box that will
allow you to reset the displayed columns L1 Fig. 8 - Edit column
to the default condition.

For this experiment we will display the

reagents which were used to stain each
sample. We would like to see the names of
the compensated parameters. To display
these keywords, in this case begin by
scrolling down to the comp FITC_Alexa
488-A reagent. Click on this parameter,
hold the shift key down to select multiple
items, and press the down arrow until all
of the reagents are selected (fig. 9). The last
one selected will be comp APC-H7-A
L1 fig. 9 - Reagents
reagent. Now press the “Add Column”
button, then press “OK” in the bottom
right-hand corner. The dialogue box will
close and your workspace will now be
populated with eight additional columns.
If you compress some of the columns by
clicking on their header and dragging
the edge of the column to the left, your
Workspace will now look like the one
shown at right (fig. 10).

The square grid to the left of

the sample name indicates that a
parameter has been compensated
during acquisition. The data was
algorithmically compensated using BD’s
FACSDiva© software. The spillover
matrix is recorded into these files as
the keyword $SPIL, so that FlowJo has L1 Fig. 10 - added columns

access to the raw data and the matrix. By

default FlowJo displays the data with the
compensation matrix applied.

A discussion on how to create or edit compensation matrices is provided in Lesson 10: Compensation.

8
The workspace now displays the list of samples and reagents.
The keywords are editable, so if a data entry mistake was
made at the time of collection it can easily be corrected in
FlowJo. Try double-clicking on one of the cells annotated
with “CD32”. The box will open and highlight the text
allowing you to overwrite it (fig. 11). Try editing the cell and
then making the correction. The only keyword that cannot
be edited directly here is the Name. You can however, select
a different keyword to be displayed under the Name column
through the preferences.

This will be explained in Lesson 11: Setting Preferences

Adding a Keyword

Click on the group “Panel_1”.

L1 Fig. 11 - edit CD32
Go to the workspace tab and
from the Keywords band,
select “Add Keyword”. When
the dialogue box appears type
“Type” into it (fig. 12). You will
get a blank column under the
header Type in your workspace.
Double-click on the top cell and
type the letter “A” into it. Data
entry can be done manually
into any of these cells, but
there are many shortcuts. With
the first T-cell selected, go to
the workspace tab and select L1 Fig. 12 - add keyword
“Copy Value to Group”. Once
you select this, you will notice
that the entire column is now
populated by the letter ‘A’. (fig. 13)

L1. Fig. 13 - keyword type

Click in the top cell in the column again, and then open the Keywords
menu and select “Create Keyword Value Series”. A dialog box will appear
allowing you to make a pattern of numbers in the column.

Click the “Keywords” tab, and you will see the interface change as
shown to the right. (fig. 14)

This tool allows you to replace selected numbers with text to create
alphanumeric patterns. Type ‘A’ in the box marked 1, ‘B’ in the box
marked ‘2’ and ‘C’ in the box marked ‘3’. Then in the bottom portion
of the interface, type ‘3’ in the box labeled “Repeat value _ times before
changing to next value”. The output, based on the values you have input,
is displayed in the bottom portion of the window in the box labeled
“Generated Series”. In this case it is A,A,A,B,B,B,C,C,C. Click “OK”. You L1 Fig. 14 - value series
will see the Type column populate with this pattern.
9
The last option for filling data into a column is through copying and
pasting from a third-party program. If you copy a column of data from
other software, such as Microsoft Excel, insert it into FlowJo by clicking
on the top cell of the column in the workspace in FlowJo that you would
like it to appear, then select “Paste” from the Edit menu.

Displaying a Graph

To display a graph of the data, double-click on A1.fcs and a plot will

appear, as shown in the figure to the right. This is a Graph Window.
There are several different kinds of plots that can be used to display flow
cytometric data. The default type is the Pseudocolor Plot. The default plot
type can be set using the Preferences dialog box (the blue heart icon in
the upper right-hand corner of the workspace). (fig. 18)

See Lesson 11: Setting Preferences for more information.

L1 fig. 18 - Graph window
The easiest way to change the graph type is to open the Options drop-
down menu below the graph image and make personal adjustments. The
drop-down menu will look like the image below. (fig. 19)

Graph Types

The graph Type menu allows you to change the graph to a contour plot, density
plot, zebra plot, pseudocolor, dot plot, histogram, or a cumulative distribution
function (CDF). An example of each plot type is displayed below. (fig. 20)

L1 fig. 20 - Graph types

L1 fig. 19 - Graph window options Left to right - top row : dot plot, histogram, zebra plot
Bottom row : contour plot, density plot, cdf (cumulative distribution function)

Contour, zebra, and density plots are useful in visual normalization of the number of collected events, as these
plots show the distribution of the data and not individual events. Contour plots show cell distribution with lines of
equivalent density that increase at user-defined intervals. Density plots do likewise with increasingly dark colors.
Zebra plots show cell distribution with a combination of lines and coloring. If rare events are important to display,
there is an option for “Show Outliers” that will plot individual dots for events outside of the distributions for these
types of plots. Pseudocolor plots, like traditional dot plots, display all events but also add brighter colors to show
increasing cell density.

10
Histograms and CDF’s (which display the data cumulatively versus increasing in number) are useful for displaying
univariate data.

Additional features within the Options drop-down menu allow you to smooth the display, change the background
or foreground colors, change from high to low resolution and make other plot-type dependent modifications.

Three-Dimensional (3D) Polychromatic Plots

To view data in three-dimensions, click on the cube icon

in the graph window.

The 3-D viewer platform will appear with a menu allowing you
to display three parameters on a cubic coordinate plane. This
allows for three-dimensional plots that convey information on
five parameters simultaneously. (fig. 21)
L1 Fig. 21 - 3D viewer

Using the drop-down menu in the top left portion of the window
allows you to select the type of 3D plot. The default is to display
each cell as a dot, or point cloud. There are also bubble plots
and terrain plots. Bubble plots show the sampled cells as larger
bubbles instead of dots and terrain plots are 2D Histograms
with number of events extruded as the third dimension. Try
each type, then select “Bubble Plot”. The figure at right shows
the 3D viewer platform with a bubble plot selected. (fig. 22)

The size of the bubbles can be set to correlate to the staining

intensity of a given parameter, allowing the user to display
a seventh parameter. Other control buttons at the top of the
screen are: Save as image, unshow axis Labels, remove the
Cube and Reset the axis to the default.

The next controls, shown below, are the

assignment boxes for each axis. When
you right-click on one of the axes, a
menu appears allowing you to assign a
parameter to a specific axis. (fig. 23)

To turn the cube or move the cube L1 Fig.22 - 3D bubble plot

around to see other populations,
simply click and hold down the mouse
button while you drag the mouse left
and right or up and down. To set the
cube in a spinning motion, click and
drag quickly left or right and the cube
will rotate on it’s own. To stop the
cube from rotating, click anywhere
on the cube. Close the 3D viewer to
continue exploring data display.
L1 Fig.23 - 3D axes menu

11
Adjusting the Scale

Double-click on file B1.fcs from Panel 1.2 and change the

parameters displayed on the axis to CD8 versus CD4. Your plot
will look like the figure to the right. (fig. 24/25) Observe that a good
portion of the data is compressed onto the axis. You will want to
adjust the scale to get all of the data on screen.

To do so, press the button on either axis to access this menu.

Clicking the “Customize Axis” option allows you to change

the axis from logarithmic to linear or biexponential. The
biexponential scale is effectively a hybrid of a linear and
logarithmic scale. The low end of the data is displayed with
a linear scale allowing negative numbers and zero to be
displayed, while the high end of the data is displayed using a
logarithmic scale. This allows the very brightly stained cells to
be compressed into a reasonable portion of the plot and viewed
as a coherent population.
L1 Fig.24/25 - CD4 vs CD8
For more information on the details of the biexponential transform, visit: http://www.flowjo.com/vX/en/gw.transform.overview.html

This data is already shown on a biexponential scale because it was collected using a biexponential scale which
FlowJo was able to gather from the FCS file. (fig. 26)

There are two sliders in this window that allow the user to modify the aspects of biexponential transform. From
left to right, you can extend the width basis and change the decade range. There are also +/- buttons to control the
positive decade range.
• Extra Negative Decades - On a
biexponential scale, some amount of
negative space is shown. If you would
simply like to show more negative space,
then you can move the slider toward 1,
adding up to one full additional negative
decade to be shown. Move the slider to
see how it changes the data display and
set it to one negative decade.

• Width Basis - The width basis is the

amount of space shown on a linear scale,
on both sides of zero. This number will
be negative to make the mathematics
of the biexponential transform work,
but you can interpret it as a magnitude.
For example, if you set the width basis
to -10 then the space from -10 to +10
will be shown linearly and the space
above and below that will be scaled
logarithmically. Move the width basis
slider up to see the effect on the data. L1 fig.26 - Custom axis control
Settle on a width basis of -398.

• Positive Decade Range (+/- buttons) - This setting allows you to control how many decades the data is displayed over.
Click the buttons to see the effect. Click Apply.(for this data file, the default Positive Decades are set to 4.42)

12
FlowJo can also help you determine the optimum width basis automatically using the “Calculate Width Basis” button.
In addition, you can manually type in your width basis allowance. Once you have set your choices you can apply
them to this particular parameter using the “Apply” button in the bottom right-hand corner of the platform. You can
modify additional parameters by clicking the vertical “Parameters” sidebar menu. The transform you have set will
be applied to each parameter selected from the list. Choose the CD8 and CD4 parameters and click “Apply” and the
window will close automatically. You will notice that the scale indicator at the bottom of your axis now reads “-1.00
+4.42/-398.00”. This means that FlowJo has added an extra negative decade, expanded the display area to 4.42 decades
and added a width basis of -398.00. Your plot will look like the figure displayed below on the right. (fig. 27B)

L1 fig.27A - Before parameters L1 fig.27B - After transformation

It is important to emphasize that using a biexponential scale does not change the data. It simply changes the
amount of space on the screen allotted to displaying various portions of your data. Statistics calculated on the
data before and after transform will be the same. However, gates made on data before a transformation is applied
may not translate between axes transformations. It is wise to look at your data, adjust the transforms so that data
is visible and then begin gating.

This is the end of the lesson on Workspaces and the display in FlowJo.

To access the Workspace at this point of the analysis, open the Lesson_1.wsp file.

•••

13
Lesson 2 : Gating and Statistics
In this lesson, you will learn how to Gate your data to create subsets and isolate a given population of cells for further
analysis. You will also learn how to calculate a variety of statistics. Subsets in FlowJo are exactly like subsets in biology:
they represent a fraction of the entire collection with specified properties (e.g., “Lymphocytes” are those cells with
low Forward and Side Scatter). You will always be asked to name a subset; the name you choose is important for
organization within FlowJo. Select a name that is meaningful to you, to help keep track of your analyses.

Creating a Polygon Gate

Double-click on the first file in the workspace to open a graph

window and change the graph to Forward vs. Side-Scatter
(FSC-A vs. SSC-A) with a Contour Plot. You will now create
a lymphocyte gate using a polygon. To do so, select the
Polygon Tool from the top of the window, click inside the graph
and move the mouse. Each time you want to place a vertex
and continue, click and continue to move. Attempt to create a
gate similar to the figure shown on the right. If you hold down
the shift key, you get horizontal, vertical, or 45º angled lines.
To finish and close the gate, either click on the starting point
or double-click at any time. As soon as you close the polygon,
FlowJo asks you to name the subset that you have just gated.
FlowJo provides a default name; however, you should choose
a name that describes the population that you gated. Name
this gate “Lymphocytes”. To accept the gate, click on the “OK”
button. If you click on the “Display” button, FlowJo will create
the gate, and open a new graph window showing only the events
contained within the gate you just created.

You will note some changes to the graph window. The L2 fig.1 - Lymphocyte gate
“Statistics” tab at the bottom of the window displays
the fraction of events in the gate. “Active Gate” refers to
whichever gate is selected. When you create a new gate it is
automatically selected. You can tell that a gate is the active
gate because the black vertices reappear on it. There is also
a new entry in the workspace window - Lymphocytes as a
subset of all of the events in A1.fcs as shown to the right.

This subset is represented by new row indented one level

below the sample which represents the subpopulation L2 fig.2 - Lymphocyte subpopulation
defined by the gate. The row begins with a subset icon,
followed by the your subset name. To the right, you will find
the frequency of these events (within the sample) and the
total number of events in this gate. Note that anything that
can be done to a sample can also be done to a subpopulation.

14
Double-click on the “Lymphocytes” subset to open its graph,
which will allow you to gate within that subset. You will see
the graph to the right. This is the contour of the events within
the lymphocyte gate. Note that the Up arrow button is now
active. Clicking on this button opens the “parent” graph, the
plot you used to create this subset. The Up arrow can be used
to navigate to the parent population.

Change this “child” graph to a CD14 versus CD3, and make

it a Pseudocolor plot; your graph will now look like the one at
the right. Note that most of the events are CD3-positive and
CD14-negative (i.e. T-cells). This is exactly what we expect
for Lymphocytes in PBMC. Click on the Up arrow button
to make the parent population visible, and then move this
window to the side so that you can see both the graph of the
parent and child populations simultaneously. FlowJo uses
dynamic recalculation of gates. This means that whenever
you adjust a gate, FlowJo automatically updates all visible
windows to reflect that change. L2 fig.3 - Graph window lymphocyte subpopulation

Editing a Gate
L2 fig.4/5 - Graph window
Move the mouse over the Lymphocyte gate in the parent parent and
graph window; the cursor changes to a four-way arrow. You Subpopulation
can click and drag to move the gate. Adjust the gate to rest
on the monocyte population as shown. FlowJo instantly
updates the graph of the subset; that window will now look
like the figure below. The new gate includes few events that
are CD3-positive and a large fraction that are CD14-positive
(i.e. monocytes).

You may continue to move the gate

around, and see exactly how the gating
affects the subpopulations. There are a
number of other ways to modify a gate.
Clicking on any vertex allows you to
drag the vertex to a new location and
change the gate shape. Selecting a gate
and pressing an arrow key allows for
incremental movement. Adjustments
to the parent graph will cause the child
graphs to automatically update.

L2 fig.7 - Lymphocyte graph

L2 fig.6 - Graph window monocyte gate window update

15
Creating a Gate on a Histogram

Move the gate back to the Lymphocyte population to reflect

the Lymphocyte cells. Click on the down arrow to return to the
Lymphocyte-only graph window. Using the Y-axis parameter
menu, select “Histogram”. The graph should now appear as
shown in the figure on the right.

You will notice that when you switch to a Histogram, two

additional gate choices appear in the icon menu at top: a range
gate and pair of range gates that bifurcate the data.

Creating a gate on a Histogram is the same as creating

a gate on a 2-dimensional (bivariate) plot. Select the
Range icon, then click on the Histogram image and drag in
either direction. Make a gate to include only the CD3 positive
cells by clicking on one side of the peak and dragging to the
other side. When you let go of the mouse, FlowJo again will ask
to name this new subpopulation. Type “T-cells” for the subset L2 Fig.8 - Histogram
name.

Bifurcating using a bisector

gate is useful for gating
the positive and negative
population simultaneously.
You only need to click once
on the graph window and
a pair of gates will appear,
with their intersection
located where you clicked.

The graph window now

displays the gate and
the statistics within it.
See figure 9 at right. The
vertical placement of the
gate is irrelevant; you can
drag the gate horizontally
or vertically by clicking
on the horizontal line and
moving it around. You can L2 Fig.9/10 - range gate L2.Fig.11 - bisector gate
change the upper or lower
boundaries by clicking on one of the handles and moving it.
You can also move the gate name and statistics independently
by clicking on them and dragging.
L2 Fig.12 - workspace T-cells
The workspace window now has a new entry, shown in the example at right (fig.12).
Note that the new entry is further indented below Lymphocytes. This is because the new subset is a subset of
Lymphocytes - critically, the workspace reflects this hierarchy.
16
The numbers at right of the subpopulations indicate the fraction of events falling within the gates. Thus, 65.9% of the
sample’s events fall in the Lymphocyte gate and 84.0% of the events in the Lymphocyte gate are in the T-cell gate. But
what fraction of the total events in sample A1 is in the T-cell gate?

Adding Statistics

To calculate additional
statistical information about
your subpopulations, you can
add statistics to the subsets.
Click on the “T-cells” row so
that it is highlighted, then click
the Sigma button from the
workspace tab, then click “Add
Statistics”.

This indicates that you want

to calculate a statistic on the
currently selected subset, in
this case, T-cells. You will now
see the Add Statistics window,
as shown to the right (fig.13).
On the left side of the statistics
L2 fig.13/14 - Workspace add statistic
window is a list of the available statistics. Some of these
statistics require that you choose a parameter on which
to calculate the statistic, for example, Median, which will
calculate the median fluorescence. If you wish to compute
a specific percentile for a parameter, you will type the
percentile into the data entry box.

L2 fig.15 - Median freq. Of total

L2 fig.16 - Stats in workspace Select “Freq. of Total”, and then click on

“Add”. Then add the Median Fluorescence Intensity for CD3 by choosing “Median”, selecting CD3 from the parameter list,
and clicking “Add”. When you have finished adding statistics to this population, close the window by pressing “Close”. Once
you close the statistics window, observe the new entries in the workspace, which will resemble the figure below (fig.16).

There are also choices for All Fluorescent Parameters, All Parameters, and All Compensated Parameters. These
options are available from the Quick Statistic drop-down menus to the right of the Add Statistics button (fig.16).

Other Types of Gates

17
 FlowJo offers several other types of gates beyond polygon gates. All can be modified and interacted with as
described for the polygon gates. The gating tool bar at the top of the graph window looks like this:

The Arrow Tool is used for selecting a gate. Once selected the active gate tools apply to that gate and the gate
can be modified.

The Rectangle Gating Tool, and as you would expect, creates rectangular gates.

Quad Gating Tool allows the user to simultaneously create four abutting rectangular gates. This gate type is
useful when you are interested in the double positive
population, both single positive populations and the
negatives for a pair of parameters. Select
your Lymphocyte population, and then display
the parameters CD19 versus CD32, two markers
for B-cells. Next open the X axis transformation
button and select “Customize Axis”. Set the Extra
Neg. Decades to 0.5, then click “Apply”. Try putting
a quad gate on your plot by selecting the
quad gate icon and then clicking once on the
CD19-CD32 plot. The result should look similar to
the figure to the right (fig.17). To move the quad gate,
click at the intersection of the quad gates hold the
mouse button down and drag. The intersection point
will move with your cursor.

In addition to traditional quads, FlowJo offers:

Curly Quads and Spider Gates. Both tools can be

selected from the gating tool bar. Note: Curly Quad L2 fig.17 - Graph window quad gate
gates can be used only with compensated data.

The Elliptical Gating Tool creates elliptical gates, which similar to rectangle gates, are ideal for gating
prominent populations that do not require complex shapes to identify, as they require the fewest vertices
to define. The number of nodes corresponds to the amount of calculation FlowJo must do to identify a
subpopulation, so fewer nodes mean faster calculations of the workspace.

The Polygon Gating Tool allows you to place points around a population as described previously.

The Freehand Gating Tool allows you to create a gate by clicking on the plot, holding the left mouse button
down, and moving the cursor anywhere. When you release the mouse button the gate will close producing
an amorphous shape.

The Auto-Gating Tool. When you select this tool and drag your cursor around the graph window, FlowJo
will automatically show you a gate with borders that follow a line of equal density; they will in effect follow
a contour. To accept an auto gate, simply click when you are satisfied with its location. Alternatively, to finish an
au t o - g at e you can click and hold the mouse button, drag the cursor either away or toward the center of
the gate, and the gate will either expand or shrink respectively. The gate center remains fixed. You are no longer
guaranteed equal density, but it allows the user some flexibility.

18
To try out this tool, choose your Lymphocyte population with
the CD19 versus CD32 parameters selected. Use the “Auto
Gate” tool to Gate the double positive population and name it
“B-cells”. You can then switch the plot type to contour to see that
the gate you have created follows the pattern of the contours.

Deleting Gates

We will now remove the quad gates. Click on a quadrant

in the graph window and press the “Delete” key on your
keyboard. FlowJo will bring up a dialogue box asking if you
really want to delete this gate (and a shortcut checkbox asking
if you would always like to show this message). Agree, and
observe that the quad gates disappear from the graph window
and the workspace. From the workspace window, click on
the 3D view and press “Delete”. The node will disappear
from the workspace list, and if it had been a gate would have
disappeared from the graph window. FlowJo allows you to
delete any type of gate in this manner. L2 Fig.18 - graph window auto gate

L2 Fig.19 - workspace end of lesson 2

19
If you have followed all of the steps, your workspace will look like the figure below (fig.19).

To access the workspace at this point of the analysis, open the Lesson_2.wsp file.

Lesson 3: Copying Analyses to Other Samples

One of the basic principles of FlowJo is that virtually all analyses that you perform on a population of cells can be
applied to other populations using the “drag-and-drop” feature. Hence, when you click on a node in the workspace (i.e
a gate or statistic), and you drag it to another place in the workspace, FlowJo copies the analysis to the new location. In
this lesson you will also adjust the biexponential transform for
two parameters to improve the display of data.

You will now analyze a set of files with a different staining

combination than A01. This lesson builds on the workspace you
finished in Lesson 2. Alternatively, you can open the workspace
named “Lesson_2.wsp” to get the workspace completed in
Lesson 2. We will analyze files B1 and B2 stained with the second
combination of reagents (Panel 1.2).

Setting up an Analysis

Start by double-clicking on file B1.fcs to open a graph window

and examine a Histogram of 7-AAD by choosing 7-AAD to
appear on the x-axis and selecting “Histogram” from the y-axis.
Make a gate including 7-AAD negative events to isolate the live
cells and name this subpopulation “Live”. Double-click on that
gate to isolate those cells, and switch the parameters to FSC-A
and FSC-W. Make a rectangular gate that includes the events L3 fig.1 - Gate live
with a consistent ratio of the two parameters as shown below to
the right (fig.1), and name this population “Singlets”. Finally, let
us apply a Lymphocyte gate to these events.

Copying a Gate to Another File

You have already made a Lymphocyte gate on file A1; presumably,

this same gate should be applicable to B1. To do this, first click
on “All Samples” in the group panel so that files A1 and B1 are
both displayed. Then click on the “Lymphocytes” subset of
A1 in the workspace window, drag it down and drop it onto
the “Singlets” node of B1. FlowJo creates a new subpopulation
using the same gate you had previously created (fig.2). Note that
you can drop a population onto any level of the hierarchy, even
across groups and to apply that gate to the selected events.

You can double-click on the “B1” Lymphocyte population and

press the “Up” arrow to see how this gate lines up with the
L3 fig.2 - Gate singlets
new data. Return to the Lymphocyte population and view a
Histogram of CD3. You have already created a T-cell gate on sample A1, and we would also like to apply that to

20
sample B1. However, gates are linked to the parameters so you need to draw a different T-cell gate for sample B1.
As CD3 was labeled with APC-Ax700 on sample A1 and PE-Cy5.5 on sample B1, we cannot directly drag this
gate between samples. For this and other reasons, it is important that you carefully enter the appropriate stain
(antibody) names when you collect the samples.

Draw a new T-cell gate on the B1 Lymphocyte population,

as shown in the figure below (fig.3). Then double-click on
the T-cell population and change the parameters to CD8
versus CD4. Use a quad gate to simultaneously gate the
CD8 and CD4 single positive, double positives, and double

L3 fig.4 - Quad gate list

negatives.

Your workspace will look like the figure below (fig.4). Click on
the disclosure triangle next to B1.fcs. These triangles open
and close the views of the subset hierarchy. In the genealogical
terminology that FlowJo uses, the subpopulations created by
the quad gates are siblings, and are children of the “T-cell”
subpopulation, and grandchildren of the “Lymphocytes”
subpopulation.

Next, add a statistic to each of the four quad gates. To do

L3 fig.3 - Quad gate so, click on the “Q1: CD8+, CD4-” subpopulation so that
it becomes highlighted. Then click the “Add Statistics”
Sigma button to open the dialogue window. Choose
“Median”, and select “CD3” as the parameter, then click
“Add”. Click on “CV” and click “Add” again to include
a second statistic. Now click “Close” to close this
window. The workspace now contains two additional
nodes under the first quad gate.

Just like gates, statistic nodes can be dragged and dropped

on other subpopulations. Drag and drop both stats to the
three other quad gates. Your workspace will look like the L3 fig.5 - Added statistics
figure below (fig.5).
Copying Multiple Gates

Now, we would like to apply exactly the same gates and statistics applied to sample B1 to sample B2. We could
drag each line one-by-one to B2. However, FlowJo provides a special mechanism for copying entire analysis trees.
When you hold down the “Alt” key (PC, or “Command” key on Mac) and begin to drag, FlowJo will take the
subpopulation you clicked and all of its descendants, including statistics. You will see this occurring via the outline
that FlowJo draws, which denotes all of the analyses that you are copying.

Click on the “Live” gate, press the “Alt” key, and drag it to the “B2” row. Even if the children of the first Lymphocyte

21
gate are hidden because you closed the triangle, they will still be copied. Your workspace will now reflect the fact
that you copied fifteen different analyses (gates and statistics) with one operation.

There are more complex ways of selecting which analyses to copy. For instance, you can shift-click several analyses to drag
multiple contiguous (within the workspace) gates simultaneously, or you can control-click to select any subsets and/or
statistics to be dragged. Try selecting just the “Live”, “Singlet”, and “Lymphocyte” gates from sample B1 and dragging it to
Sample B3.

Deleting Analyses

You can delete analyses by selecting them and pressing the “Delete” key. Select the “Live” subpopulation that you
just added to B3, and press “Delete”. When you delete the Lymphocyte gate, all of the subsets of Lymphocytes
will also be deleted. This is because those subsets have no meaning without a live gate present. Remember that
every subset you name is, in reality, a subset defined by all of the ancestor populations: the parent population,
grandparent population, etc.

To access the workspace at this point of the analysis, open the Lesson_3.wsp file.

22
Lesson 4: Groups
In this lesson, you will learn how to take advantage of sample groups. Groups are the principal functional unit
within FlowJo and one of the primary mechanisms by which FlowJo allows the user to perform batch analysis; the
repetitive application of analysis or generation of reports across many samples. You may create as many groups as
you wish; each group may include any of the samples within the workspace. Samples may also belong to more than
one group. Importantly, applying a gate or statistic to a group will be equivalent to doing the same thing to every
sample in that group—the advantage is that you do it all at once.

In Lesson 3, you learned that by dragging multiple analyses or entire sets of analyses, you can accomplish the first
step in batch processing: the ability to replicate multiple analyses. But that still leaves the next step: how can these
analyses be applied to a whole set of samples at once? To accomplish this, FlowJo allows you to create groups of
samples. A group is a list of samples from the workspace that can serve as a sample surrogate. That is, you can
apply analyses to a group much as you can apply them to individual samples. There is one rule about groups and
group analyses that is inviolate: every sample in a group contains every gate, statistic, or other analysis that has
been applied to the group.

This lesson builds on the workspace you finished in Lesson 3. You may also open the pre-made workspace named
Lesson_3.wsp.

The first step is to create a group that contains a set of samples that will receive similar kinds of analyses. In
this tutorial we already have a number of groups that were created by the filing structure when we loaded data
into FlowJo that could be analyzed in the same way. Now you will make a new group that gathers all files with a
particular staining combination, regardless of the file structure.

Begin this lesson by adding “Exp. 2” to the workspace. Locate the folder with the downloaded demo data and drag
“Exp. 2” into the workspace just as you loaded Exp.1 in Lesson 1.

Creating a New Group

In the workspace, click on the “New Group” button, which can be found in the shortcut bar at the top of the
workspace, as well as the Navigate task in the FlowJo band. FlowJo brings up the Create Group window shown
below (fig.1). Using this window, there are a variety of ways to make new groups. The top portion of the window
controls the format of the group’s appearance in the workspace, and the middle portion allows you to set your
sample inclusion criteria to control which samples will be added to the group. In sample inclusion criteria, there is a
window that lists every combination of reagents present within the samples. They are ordered by collection channel.
Click on the CD56, CD11c, CD3,
CD45RO, 7AAD, 2H2, CD4, CD8
combination, telling FlowJo to
group all samples in the workspace
with this stain combination.

Type the name “T-Cell Panel” in

the name box at the top, change
the color to red by clicking on
the “color box”, and the font style
to “Plain”. Click on the “Create
Group” button, and then the
“Close” button to close the window
and display the workspace. L4 Fig.1 - new group and options
23
Each group is displayed in the group section of the
workspace, in the color and font style you have selected.
The number to the right of each group tells you how
many samples are included in each group (fig.2). Since
there were nine subjects labeled with each reagent panel
for two experiments, there are now 18 samples in your
T-cell group.

Modifying an Existing Group

Double-click on the name of the group you want to

modify. In this case, double-click on “Panel 2.1”. Rename
the group “B-Cell panel” by typing over the existing name.
Because this group was created automatically when you
brought in the data, there is no criteria already defined
for it. Click on the other panel of regents, the panel that
contains CD32, CD86, etc. Change the color to green by
clicking on the color box, then click on “Apply Changes”. L4 fig.2 - Modify group
You will notice that the name change has taken
place and there are now 18 files in the group.

Deleting a Group
To delete a group, simply click on it and press
“Delete” on your keyboard. To try this, click on
the “Panel 1.1” group and delete it. Repeat for the
“Panel 2.2” group.

Creating a Group Using Keywords

Display another keyword in the workspace. Right-
click on the header of the “Name” column in the
workspace and choose “Edit Columns”. Select the
keyword “Day” from the list on the left and click
on “Add Column”. It will appear in the list to the
right. Now select “Day” from the list on the right
L4 fig.3/4 - Add “day” keyword
and drag it to be the fourth keyword from the top
under #Cells. Click “OK” to apply this change (fig.3/4).

You will notice that the column “Day” has appeared numbered from
0 to 16. Using this keyword annotation, we can “isolate” the files that
were stained on the first eight days of the experiment. To create a
group to do so, open the “Create Group” menu again. This time you
will use a keyword to define group membership. Below the staining
panel window there are a set of dialogue boxes that allow you to select
a keyword, a qualifier and a value. From the box initially filled with
“*Any Keyword*”.

Choose the keyword “Day” from the drop down-menu. The box
to the right gives you inclusion criteria. Choose “<=” (is less than
or equal to). You can then either type a number into the next
specification box, or click on the “Choose” value box to see what
L4 fig.5 - Keyword grouping
values were entered for that particular keyword within the data.
Type or select “8”. Name the group “Day 0-8” and color it purple. Your
24
group definition window will look like the window pictured to the right (fig.5).

Click “Create Group”, and then the “Close” button to close the window and display the workspace. Notice that you

L4 fig.6 - Day 0-8 group

have isolated all of the files with days numbered eight or less.

Each group is displayed in the upper (group) panel, in the color and style that you have chosen. Your workspace
should look like this (fig.6):
Adding Analyses to All Members of a Group

Some of the files in the groups already have gates and statistics. You will now apply those analyses to the rest of the
files in each group. Remember that a group is a sample surrogate so any analyses that you apply to the group will
be applied to all of the samples in that group.

Click on the “T-cell panel” group, and expand the gating tree under file B1. Click on the “Live” gate in the lower
samples panel. Hold down the “Alt” key
1. on a PC (“Command” on Mac. Then
with the left mouse button depressed,
drag the gate and release the button on
top of the “T-Cell Panel” group in the
2. group section of the workspace.

3. Notice that several things have occurred:

• the analysis tree has been
added to a row indented beneath the group
name as if it were a sample

• the analyses have been added to

every sample in the group

• all of the analyses appear in red,

which is the same color and style that you
defined for this group

Your workspace should look like the figure L4 fig.7 - T-cell panel applied
25
above (fig 7). The color and style of the analyses are an important cue. Any analysis that appears in the workspace in
the color and style of the group is guaranteed to be exactly the same as the group’s version. Thus, the gate will be in
the exact same location, applied to the same parameters, etc. This is used to ensure that the exact same analyses have
been done on all samples.
1.
Modifying Group Gates

2. There are two scenarios in which you may wish to modify group gates:

• Adjusting a gate on an individual sample

to make it different than the rest of the

L4 Fig.8 - B-4 singlets adjusted

group

• Adjusting the gates of all samples in a

group

In the first scenario, simply open the sample that you would like to modify by double-clicking on it and then adjust
the gate. FlowJo will remove the font formatting from that particular gate to indicate that it no longer matches
the group gates. To try this, double-click on the “Live” gate for sample B4. The graph window will display FSC-A
versus FSC-W showing the gate added to identify the singlets. Click on the gate and then drag it to better match
the population. If you look back at your workspace, you will see that the “Singlets” population no longer has its
special formatting as shown in the figure to the below (fig 8).

In the second scenario, you have moved a gate and would like to apply that change to the whole group. To do
this, drag the modified gate up to the group and drop it into the proper location in the hierarchy. Imagine that

26
the modified singlets gate on B4 is a better gate
for the entire group. To apply this to the entire
group, click on that specific “Singlets” gate in the
workspace. Then drag and drop it onto the “Live”
population of the T-cell group in the group portion
of the workspace window. A box will prompt you
to replace the previous gate name. Click “Yes”. If
you use the right or left arrow keys in the graph
window, you can scroll from sample to sample and
see that they all reflect the change made.

This method could grow tedious if you modify

many gates. A shortcut to execute rapid gate changes
for entire groups is to use the “Synchronize Gates”
function. Double-click on the “T-Cell Panel”
group (from the group portion of the workspace).
The group definition window will again appear.

Notice that there are two check-boxes near the top

of the screen: one for “Live Group” and the other
for “Synchronized”. The “Live” box allows the L4 fig.9 - Synchronized checkbox
group to update as you continue to add or modify
metadata. The “Synchronized” box links the group’s gates so that if you modify a gate on one sample, that gate is
modified on all of the group’s samples. Check the “Synchronized” box, click “Apply Changes” and then “Close”.
Now double-click the “Live” gate on file B5 and adjust the “Singlets” gate (fig.9). Notice that the gates font format in
the workspace did not change, and that all of the statistics updated. This indicates that all of the gates have changed
simultaneously.

To complete the analysis of this data set click on the “B-cell panel”, and then the “Lymphocyte” population of
sample A1. Hold down the “Alt” (“Command” key for Mac) key and then drag the entire hierarchy to the “B-cell
group’ in the group panel and release the mouse button. All of the files in that group will now be gated and the
statistics will be applied to all samples. Note that statistics can be applied to groups in the same manner as gates.

Now double-click on the “Lymphocyte” population of sample A1. Move the “B-Cells” gate in the graph window
and notice that the annotation turns black in the workspace. Click on the modified gate “B-Cells” in the workspace
list and press the “Delete” key on your keyboard and click “Yes” in the confirmation dialog box. You will notice
that the B-cell gate is replaced by the B-cell groups’ color and that the gate position in the graph window is reset
to the group default.

The work to this point will be saved as Lesson_4.wsp.

Lesson 5: Tables and Collating Data Output

In this lesson, you will learn how to transfer statistical analyses to tabular form for further analysis in FlowJo or for
export to spreadsheet/statistical programs. You will be able to generate a table of statistics that brings together any
set of values from any combination of samples (using groups to define the sample list).

FlowJo provides tools to collate the output from multiple analyses so that you can import them into spreadsheets
or to a machine-readable format for statistical or mathematical packages. FlowJo is not a comprehensive data

27
presentation package. For statistical analysis beyond what FlowJo provides, you will need to export your data to
other programs.

Use your existing workspace from Lesson 4, or open the tutorial workspace Lesson_4.wsp.

Open the “Table Editor” by clicking on its icon in the shortcut bar at the top of the workspace or select the “Table
Editor” from the FlowJo band. FlowJo shows you the Table Editor window as shown below (fig.1). You can create as
many tables as you like and generate a table from as many groups as you wish.

At left in the Table Editor window you will observe the list of existing table definitions. When you press the batch
for “display” button, the statistics you have selected as columns will be applied to the current group and a table
will be generated in FlowJo. A table definition is simply a list of statistics, keywords, or formulas that will become
the columns of a table when you batch. They are listed as rows in the table editor because vertical text is difficult
to read. Each row in the template defines one statistic to export such as frequency, mean fluorescence of
FITC, etc. When you create the table, FlowJo cycles through each sample in the current group and requests
the particular statistic defined in the table.

Creating a New Table

At this point it is useful to arrange

your computer screen with the
workspace and the table editor side-
by-side. This will allow easy addition of
statistics to the table editor by directly
dragging either statistics or populations
L5 fig.1 - Table editor
from the workspace into the table editor.
Start by naming your table so that you can differentiate it from other tables you may create. In the Name box at the
top of the window, double-click the table named “Untitled” and type “T-Cell table”.

Go back to your workspace and select the “T-Cell Panel” group. We will use keywords to help define the order of the
table. To do so, we will extend the keywords to the entire
T-cell panel group. Go to the workspace tab, click the
“Keywords” drop-down menu and select “Add Keyword”.
Click on the top cell in the “Type” keyword column, then
select “Create Keyword Value Series” from the Keywords
menu. Enter “3” in the “Repeat Value” box. Then click on
the “Keyword” tab and enter the letters “A” through “F” in
the six boxes (fig.2).

Click “OK”, and you will have a value series of these

letters under the keyword “Type”. The resulting pattern
in the workspace is displayed in the figure below (fig.3).

Still in the “T-Cell Panel” group, select the “Live”

subpopulation from sample B1, hold down the “Alt”
key (or “Command” on Mac), and drag the highlighted
rows into the bottom portion of the table editor window.

The table editor will look like the figure below. L5 fig.2 - Keyword value series

By using the Alt (or command) key, you were able to

28
select all of the subpopulations and statistics downstream of the Live cells. You can of course select one population
or statistic at a time if you so desire. The default is to display the frequency of the parent for each of the populations
that were added. To change the statistic,
double-click on the row that you would like
to modify.

Once the data is dragged into the table,

select the first two rows of “Live” and
“Live/Singlets” and press “Delete” on
your keyboard, clicking “Yes” to accept

L5 fig.3 - Workspace value series

the deletion. Next, move the “lymphocytes/T-cells”

population above the Lymphocyte (Live/Singlets/
Lymphocytes) row by selecting, dragging and dropping
to reorder the table.

With the “Lymphocyte/T-cell” row selected, click on

the “Edit” tab, then the “Edit Column” button. The Column Information dialog box will appear. In the column
heading, type “#T-cells”. Select the “Count” in the left hand menu, and then click “OK” (fig.5).

Now we will edit the statistic for Lymphocyte population using the Column information dialog box, as above.
Click on the “Lymphocytes” row (live/singlets/lymphocytes) to edit it similarly, type “#lymphs” into the heading,
and select “Count” from the statistic menu and click “OK”.

Now that we have the two starting populations, we can begin organizing the populations within the table. Select the
Q1, Q2, Q3 and Q4 Frequency
of Parent statistic, and drag and
drop them into ordered place
below one another so that they
are ordered one through four.

Now we will do the same for

the (robust) CV for populations
Q1 through Q4. Once
Lymphocytes and Frequency of
Parent are organized, Median
is automatically ordered below L5 fig.5 - Table editoredit column window

29
the first two.

At this point, the table columns should be named so that we can keep track of our statistics. Double-click on the
first empty row of the Name column, and type names into the rows as shown in the figure below (fig.6).

Adding a Keyword or Formula to a Table

In the Edit tab, click on the “Add Column” button and the column information dialog box will again appear. This time select
the “Keyword” tab. When you select “Keyword”, the menu will change to show a list of all keywords. Pick “Day” from the

L5 fig.6 - Table editor nameing list

list, then click “OK”. Repeat this process to add the keyword “Type,” the keyword that we added earlier in this lesson.

Next, we will add another column - but this time we will create a Formula by opening the “Add Column” menu and
selecting the “Formula” tab instead of the Keyword tab. Name the formula in the column heading by typing “Ratio -
Lymph/T-cells”. Then click on the “Insert References” drop-down menu and select “#Lymphs” (fig.7/8). Now click on
the divide symbol which will create the ratio after we add the “#T-cells. See the figure on the next page (fig.9, on next page)
for what your table will look like at this point.

You may use this interface to add any statistic

that is not a basic MFI (median fluorescence
intensity), count, etc. You can do so by choosing
information that is already in the table using by the
“Insert Reference” box, and modifying it with an
assortment of functions selected from either the
shortcut buttons or “Insert Function” box. Use this
tool to create a ratio of Lymphocytes to T-cells.
L5 fig.7/8 - Adding key word or formula
Now the table is ready to be batched.

Batching to Create a Table

30
Within the table editor tab, there are L5 fig.9 - Table editor batching options for iterations and
outputs. In this case, simply press the batch to display icon at the top-right of the table editor.

The group that you will draw the samples from must be specified, as well as the iteration, which is the order and
pattern in which the files will be organized. This is accomplished in the group drop-down menu from the Iteration
ribbon (fig.10). The default selection is the group that is currently highlighted in the workspace window, which
is the group from which you selected the statistics and populations. If you wish to apply the selected statistics to
another group, click on the drop-down menu next to the word “Group”, and select the desired group. For this
experiment, accept the default condition of T-cell panel.

Table Iteration
Iterate by Sample means that you will include every sample
in the selected group. Create a table by choosing the “T-Cell
Panel” as the group, and Iterate by “sample”. You will generate
the table below (fig.11). L5 fig.10 - Table editor groups
FlowJo cycled through every sample in the chosen group, calculated
the 11 requested statistics, created one formula and included two keywords. If a sample did not have the subset or keyword
requested there would be a blank entry in the table.

31
Iterate by Panel means that the files are organized into a panel, or set, that have some relation to each other. In this case
you may only want to include a specific member of each set, and iterating by panel will allow you to do this. Once you
set “Interate by” to “Panel,” you must fill in a value for the Number of Tubes box. This number tells FlowJo to include one
tube out of the entered number of tubes in your table. For example, if you change the “1” to “2”, FlowJo will include one

L5 fig.11- Batched table

out of every two tubes in the generated table.

Create a table that includes one file from each “Type” by selecting Iterate by Panel and entering the value “3” into the
Number of Tubes / Panel data entry box (fig.12). This will create a table using only samples B1, B4, B7, D1, D4, and D7.

Iterate by Keyword means that FlowJo will use sample keywords to determine the order and inclusion of files which
generate the statistics in the table. The order of the samples in the table will be based on the iteration keyword.
Which files are used in the creating the table is based on the discriminator keyword. Any sample with a keyword
entry matching the keyword entry of the discriminator keyword of the first sample in the chosen group is included.

In the Iteration task, choose “Iterate by keyword” and press the key icon which will open a window allowing you to
choose what keywords you would like to “Iterate By” and
which keyword will serve as the “Discriminator.” Choose
iterate by “Day” and discriminate by “Type”, then press
“OK” (fig.13). The resulting table will include only the first
three samples, because they are the only samples that
contain the letter “A” for the keyword “Type”.

If you iterate by “Type” instead and discriminate by “Day”

your output table would include only samples B1 and D1
(fig.14). L5 Fig.12- iterate by keyword

Additional Tools

Returning to the table editor window, there are several options that have not
yet been discussed. (fig.15)

The ( and ) buttons allow you can create new table templates, duplicate
existing table templates, or delete existing table templates respectively. The

L5 fig.13- Keywords

L5 fig.14 - Table results

“to file” button icon allows you to batch a table to file.
32
The drop-down menus below that allow you to specify the group and the iteration options for the table you will
create. In addition you can batch a table to the printer or to the current layout.

L5 fig.15 - Table editor shortcut keys

Add a second table by clicking the button in the table editor window.
Type “B-Cell Table” into the name box. From the workspace, select the “B-Cell Panel” group. Open
sample “A1” and select the “Lymphocyte” population. Hold down the “Alt” (or “Command”) key and drag the selected
populations into the table editor. Add a formula by clicking on the “Add Column” button.
Choose the “B-Cell Count”, type a “/” (division symbol), then choose the “T-Cell Count” and
give the formula the name “BT Ratio”. Click “OK”. If you add custom names to the statistics and change the
“Frequency of Parent” statistics to “Counts”, your table editor will look like the figure below at left (fig.16).

Applying Conditional Formatting

Click on the heart icon in the upper right hand corner of the table editor window. This will open FlowJo preferences.
Under “Tools”, click to open “Ranges”.
Within Ranges you are able to set any
conditional formatting you would like to
reference in your analysis. You are
able to enter the target population
with a minimum and maximum allowance
(fig.17). Once all entries are complete, click
“OK” to close preferences. Back in the table

L5 fig.16 - Table editor updated

editor, highlight the statistic to which you would like to apply
formatting. Select the “Column” tab and click “Expected Range”
in the Formatting band. In the drop-down box you can select
the specific population to view.

Dynamic Updating

The table window is always current. When you create a table,

FlowJo goes through all the samples and makes sure they
are recalculated according to the latest L5 fig.17 - Range batch settings
modifications. If you now go back and change the Lymphocyte gate and apply that change to all of the samples,
that change will be immediately reflected in the table window.

The work to this point will be saved as Lesson_5.wsp.

Lesson 6: Creating Simple Graphical Layouts
33
This lesson describes the fundamentals of the Layout Editor. You will be able to generate layouts with multiple
graphics, statistics, etc. and learn how to create overlays of graphs. In Lesson 7, you will learn how to create batch
reports, where the layout can be generated automatically from all of the samples in a group. Lesson 8 demonstrates
the features in the Layout Editor that allow creation of complex multi-sample layouts.

This lesson continues with the workspace document you finished in Lesson 5; alternatively, you can open the
tutorial workspace named “Lesson_5.wsp”.

Creating a New Layout

To open the Layout Editor, either press “Ctrl-L” (“Command-L” on a Mac), or click on the “Layout Editor” Icon.

In the Layout Editor window, the upper left portion of the window has a list of all of the layouts you have created i n
this workspace. You can have as many
layouts as you wish. This portion of the
layout editor works just as in the table
editor does. You can name the layout
by clicking in the name field “Untitled”
near the top of the window and typing
a new name. Give this layout the name
“B-Cell plots”.

Click on the top-level (ungated)

population of sample A1 from the
B-cell panel group. Hold the mouse
button down and drag it into the
“Layout View”. If you double-click on
the plot in the layout editor, a dialog
box (the Graph Definition window, fig
3, below right) will appear allowing you
to make changes to the graph. Switch
it from contour to pseudocolor. The
result should look like the image to
the right (fig.1/2). The default graph is
the parameter combination and plot
type that was last viewed. FlowJo also
creates an annotation text box below L6 Fig.1/2 - layout editor with tools
the graphic that contains pertinent
information.

Any graphic item can be resized

L6 Fig.3 - graph definition window
or moved simply by clicking and
dragging. To resize, click and drag on one of the four handles at each corner. You can also change the magnification
of the view by clicking on the scaling tool, the magnifying glass in the bottom left hand corner of the screen.

One of the important aspects of the layout editor is that it is live. This means that any time you change or move a gate
or modify an analysis in the graph window, FlowJo will automatically update the layout editor if needed. Thus, you
can use the layout editor to provide instantaneous feedback for gating operations, where you can simultaneously view
many different subsets (even multiple views of the same subset) while moving a gate used to define that subset.

Creating a Second Graph of a Population 34

To create another view of the same subset, you have three choices: (1) drag the same subset from the workspace window
into the layout editor again; (2) select the first graph in the layout editor, right click and do a “Copy” and “Paste” operation;
(3) hold down “Alt” (PC, “Option” on Mac) key, click on the graph to be duplicated and drag this duplicate to a new
place. For now, duplicate the first graph using the copy/paste method and place the copy next to the original.

Editing a Graphs Appearance

To change how a graph looks, double-click on it; the graph

definition dialog appears, as shown to the right. From this window,
you can specify exactly how you want the graphic to appear.
Switch the parameters to CD3 versus CD14, from the X-Axis and
Y-Axis boxes. Change the graph type to Density, and check “Show
Outliers”. You will need to deselect “smoothing” if checked.

Click on the “Annotate” tab at the top of the window. This shows
a different set of options that control what appears in the graphic,
such as gates, frequencies, legends, adjunct Histograms, multigraph
overlays, backgating, etc. Deselect the Annotation checkbox (fig.3).
This will remove the annotation text below the graphic. Click “OK”,
and the layout editor will look like the figure below (fig.4).
Aligning Plots

L6 fig.3 - Graph definition window

FlowJo
L6 fig.4 - Layout editor after changes
provides many features of drawing programs, such as the ability to
align multiple objects. To align these graphs, left-click the mouse and
with the button depressed, drag a rectangle to include both graphs.
This selects the images for formatting with the Marquee Tool (fig.5).
Now from the Arrange drop-down menu, click “Top” in the Vertical
Alignment options. Notice that there are also selections for horizontal
alignment, equal spacing and spread.

Dragging Statistics into the Layout Editor

L6 fig.5 - Layout editor align tools

35
To display a statistic that you have added to the workspace, you can drag and drop it just like dragging of gated
populations. To try this, click on the “Median: CD3” of the Lymphocyte population from sample A1 and drop it below
your first plot. If you would like to format or edit the text, double-click on the statistic once to activate the edit, then
again on either the name or number and the edit box will appear, as shown to the below (fig.6). The information within
the angle brackets < > is the calculation of the statistic. You can change anything else without affecting the statistic.

Creating Formulas in the Layout Editor

In the workspace, add the statistic “Count” to both the

B-Cell and T-Cell populations of Sample A1, using
the Statistic band (sigma icon) within the workspace
panel menu, as explained in Lesson 2. Then drag the
two counts to the B-cell Panel group. Next drag the two
counts separately from Sample A1 into the layout editor
and place them below the figure. Double-click on the
number portion of “T-Cell Count”.

Now click “cancel” the FJML editor box.

Imagine that you want to create an additional statistic,

the sum of the T-Cell and B-Cell counts. The easiest
way to do so is duplicate one of the existing statistics
and edit it to show the desired result. Click on the
“B-Cell Count”, and duplicate it by using the keyboard
shortcuts “Ctrl-C” and “Ctrl-V” to copy and paste it
into the layout (fig.7).

Next, L6 fig.6 - Layout editor text fjml box double-click

L6 fig.7 - Layout editor copy & paste

on the number portion of the “T-Cell” statistic. It will
look like the text box at right (fig.8).

L6 fig.8 - Layout editor t-cell text edit

36
As you can see, “T-Cell Count” is outside of the angle brackets. This
portion of the annotation is simple text that you can edit to say whatever
you like. The statistic portion of the annotation is calculated only within
the brackets. Information within the brackets provides FlowJo with: (1)
which file the statistic is from, (2) the gating path and (3) the particular
statistic to be calculated.

Highlight all of the information (from bracket to bracket) and copy it

using the keyboard shortcut “Ctrl-C”. Click “OK”. Activate the FJML
editor on the number portion of the duplicate “B-Cell Count”. At the
end of the statistic, (outside of the angle bracket) type an addition (+)
sign. Then use the keyboard shortcut “Ctrl-V” to paste in the statistic
you copied from the T-Cells count after the addition sign.
L6 fig.9 - Text editor formula
To tell FlowJo the extent of the formula, type in the information in the figure to the above (fig.9).

Always use double curly brackets {{ statistic }} to encapsulate the statistic you wish to apply. To add a title for the statistic,
type “B-cells+T-cells =” before first curly bracket. When creating the formula, type a title outside of the angle brackets
(and curly brackets if present). The title on the formula should now read Click “OK” to accept the modifications.

Drawing Tools

FlowJo’s layout editor provides a few simple drawing tools to embellish your graphical reports. These tools can be
selected by clicking on one of the icons on the top-left portion of the window.

You can choose :

The Arrow Tool is used to select existing objects. Click on an object to select it (as shown by dark handles at
the corners of the object). Use the “Shift” key to select additional items. Drag items to move them. “Alt”-drag
(or “Command”on Mac) to duplicate them. Start a drag in the background of the layout editor, and it will
draw out a rectangle. Upon finishing the drag, the layout editor will select all objects that are enclosed by that
rectangle. (Marquee selection.)

The Rectangle tool is used for drawing simple boxes and frames. If you create the rectangle surrounding
another element, and want it to serve as a background, go to the Arrange section of the ribbon, and select use
the Arrange > “Send To Back” menu command to change the order of the layout elements you have selected.

The Line Tool is used for drawing lines and arrows. Generally, you will want lines drawn on top of other
elements, so draw them last or use the Arrange > “Bring To Front” command to change the order of the
layout. Right-click on the line and select “Arrow Style” to add arrowheads to the line.

The Grid Tool will create a matrix of cells within your layout. Each cell may contain text, charts, images, or
other grids. The grid provides a convenient mechanism to group and align multiple elements of the layout.
Grids are explained in greater detail on the Grid Tool page.

The Text Tool is used for adding textual annotation to the layout. To create a text box, you select the text tool
from the toolbar, and click once or drag out a rectangle in the layout view. When you create a new text box, a
dialog will appear to help you edit the text. This is called the FJML (FlowJo Markup Language) Editor. FlowJo
uses a custom markup language, similar to the HTML used in web pages, to richly express the structure
of your flow analysis. Once you confirm changes via the mouse or the Enter key, or click the mouse on a
different object in the layout, the editing stops, and the layout editor text is reformatted and frozen. To edit it
again, double-click on the text box to return it to the edit state. Text clippings or statistics from the workspace
can be dragged and dropped into a layout. In both of those cases, a text box is created automatically.

37
 The Ellipse tool is used for drawing simple circles and ellipses. If you create the ellipse surrounding another
element, and want it to serve as a background, use the Arrange> “Send To Back” menu command to change
the order of the layout.

The rounded Rectangle tool is used for drawing simple boxes and frames. You can drag the dot to change
the overall shape. If you create the rounded rectangle surrounding another element, and want it to serve as a
background, use the “Send To Back” button under the Arrange menu to change the order of the layout.

The Diamond tool is used for drawing quadrilaterals that point to the center of each bounding edge. If you
create a diamond surrounding another element, and want it to serve as a background, use the “Send To Back”
button under the Arrange menu to change the order of the layout.

The Triangle tool is used for drawing 3-sided shapes in your layout report. If you create a triangle surrounding
another element, and want it to serve as a background, use the “Send To Back” button under the Arrange
menu to change the order of the layout.

The Statistics Table Tool allows you to build a table of statistics within the Layout editor. Click the statistics
button, then drag a rectangle in the area where you want to place the statistical table. A statistics table dialog
box will appear allowing you to enter data.

The Web Box Tool creates a box with an embedded html viewer so you can display parts or all of a web page within your layout.

To add text to your plot, click on the Text button and draw a rectangle that will contain the text. Type the
word “Sample”, then right-click and select “Insert Keyword”. Choose the sample “A1”, then choose the keyword
“Tube Name”. Click “OK”. Now with your text selected and highlighted, go to the Object tab at top of the layout
editor. Change the font size to 18 using the font drop-down menu in order to make it easier to read. You will notice
that the text “Sample A1” has appeared on the plot where you placed the text box. FlowJo has filled in the value for
the selected keyword and placed it in line with the rest of the typed text. Using keywords instead of typing all of the
text will be important in Chapter 7 when you create batched plots for all samples in a group.

Overlays

In the layout editor tab, click the

on the upper left to add a new
blank layout. Title this layout
“Overlays”. Select the Lymphocyte
population from sample A1 and
drag it into the layout editor. Any
graphic item can be made into an
overlay by dragging and dropping
another subset onto it. You can
overlay different subsets from the
same sample, or overlay plots from
different samples. For now, select
the “T-cells” subset from the same
sample and drop it on the graphic
(the sample name will appear in
the legend to show that is was
dropped). Then drag the B-cells
L6 fig.10 - Layout editor overlays
subset and drop it on the plot as
well. You should now see the multi-
color Dot Plot, shown here to the right (fig.10). 38
FlowJo draws two Dot Plots, one for each subset, in the same graph. In addition, it automatically creates a legend
for the overlay, shown to the right of the graphic. To make changes to the legend, double-click on it. Click on
the “Sigma” button and add the MFI of CD3 to the legend by choosing it from the pop-up menu. Once you have
selected the CD3 Median, click “Add” and then “Close”. Click “OK” in the graph definition dialog box. You can
change the color of any subset by clicking on the color box next to that subset in the legend.

You can change the stacking order of the colored layers by clicking on any item, and dragging it up or down in the
legend list. Finally, you can delete an item from the overlay by right-clicking and selecting “Remove Layer” from
the pop up menu that appears. You can overlay an almost unlimited number of different subsets on the same graph.

Note: you can choose to show the Legend box for any graphic, even if it is not an overlay, by double-clicking on a
graph to open the Graph Definition Window and access Annotation options. There, you can also set color and line
styles for single graphs just as you can for overlay graphs.

Once you create an overlay graphic, you can easily change its appearance (axes and plot style) – just like any
other graphic. For example, double-click on the “Overlay Plot”, choose the “Specify” tab, and change the Y-axis to
Histogram. Click “Apply”, then “OK”.

If you right-click on any sample in the legend, you can select the line weights (Hairline, Normal, Heavy, or Very Heavy)
the line styles (None, Solid, Dotted, Dashed, Long-Dashed, Complex, or Dot-dashed) or if the Histogram coloring
(None, Filled or Tinted). You can also remove one of the overlaid populations, or use this interface to edit what is
displayed in the legend. Fill in the B-Cell layer. Now hold down the “Alt” key (“Command” on Mac), then Right click on
the “T-Cell” layer and select “tinted 80%”. Notice that all layers follow the format command if the “Alt” key is depressed.

Ancestry and Backgating

Create another new layout using the button, and call it “Backgating Plots”. This time, drag in the “T-Cell” population
from sample A1. Right-click the plot and you will see the menu displayed
to the below left (fig.11).

Most of the graph options can be selected from this interface. Click
“Ancestry”. You will notice that your graph now includes two smaller plots

L6 Fig.11- Layout editor menu

L6 fig.12- Backgating ancestry

39
that illustrate the strategy used to isolate the events within the
T-Cell gate. The resulting plot is shown below (fig.12).

Right-click on the graph again, and select “Backgating”. The

ancestry plots now show the back-propagation of events from
one gate to the next. This is a tool for evaluating the effect of
any gate. The result is shown in the plot on the right.

Multigraph Overlays

FlowJo offers a convenient feature for viewing multidimensional

data using many parameters simultaneously called a Multigraph
Overlay. Below the ancestry plot, drag the “T-Cell” population in
again. Set the graph to be “CD3” versus “SSC-A” as a smoothed
L6 fig.13- Backgating
pseudocolor plot. Right-click on the plot to open up the pop-
up menu. The bottom choice is “Make Multigraph Overlays”.
When moused over, it expands to show three choices. Select the
top choice: “Histograms”.

FlowJo will display the Histogram of the plotted cell population

(the T-cells in this case) for all parameters. The resulting plot is
shown above (fig.13).

If you right-click on the plot again, you can hide the multigraph

L6 fig.14 - Multigraph overlay

overlay, and then select the second option (“All Parameters

by Y”) to show all plots by the parameter on the Y-axis, in
this case SSC-A (fig.14). Select this and you will get a plot with L6 fig.15 - All parameters by y
SSC-A on the Y-axis, and the rest of the parameters shown
on the X-axis one at a time. A portion of the resulting figure
is shown below (fig.15).

If you hide the multigraph overlay again, then switch to the

final choice. NxN, FlowJo will make a grid of all possible
two-parameter combinations. Additionally, if you overlay
another population on the original graph, the additional
population (or populations) will be overlaid in all of the
NxN plots. Try dragging the “B-Cell” population onto the L6 fig.16 - NxN plots/combination
original T-Cell plot. The resulting figure is shown to the
right (fig.16).

Displaying Adjunct Histograms

Choose to hide the multigraph overlay. Right-click L6fig.17 - Adjuct histograms

40
on the plot again and this time select “Adjunct Histograms” from the pop-up menu. This selection will add a
Histogram of each of the component parameters in a two-dimensional plot to the axes. If you have an overlay (as
we do in the case), FlowJo will display the histogram of each population overlaid in the histograms as well. The
resulting graphic is shown to the right (fig.17).

L6 fig.18 - Export options

Exporting a Layout Page

The tools at the far right-side of the toolbar, shown below (fig.18), allow you to export a layout page to some other
format. They apply to the layout page that is open.

The result of a batch report can be viewed in several different ways. Clicking the “Batch” button can create:

• A new layout in the layout editor.

• Alternatively, it can print the report directly to your printer.

• A web page containing the pictures for each layout group

• With web animation, an HTML file will be created that contains an embedded Quicktime movie with options.

• With animation, each of your batches will become a continuous frame in a movie format.

• Each page of your batch will become a slide in PowerPoint.

• A preview window containing static tiles-PDF (data will not update if you change the original analysis).

In every case, FlowJo will produce a series of frames, each one containing graphs and statistics from one
or more tubes.

Printing from the Layout Editor

The printer icon in the File tab opens a menu allowing you to choose a printer to print a hardcopy of the current
layout. You will notice that FlowJo draws gray dotted lines on the Layout Editor pages: these correspond to page
breaks in a printed document. (Select a small magnification, like 12.5%, to see many pages at once). Note that as
you change the viewing magnification, the relative scaling of the graphs to the page boundaries do not change –
you are not changing the print magnification! You can change
the relative scaling of all graphs by clicking and holding on an
intersection point of the gray lines, then either dragging away
from the origin point to stretch all of the pages, or dragging
toward the origin point to shrink all of the pages. By using this
method to scale plots, all plots will always remain the same
L6 fig.19 - File panel options size. Additionally, the orientation of the pages can be toggled
between landscape and portrait, by grabbing an intersection point and dragging upward diagonally.

There are also shortcuts that allow you to specify that FlowJo automatically scale all plots to fit on one page to be a
41
certain width, height, or one full page. This is done by selecting “Scale to Page Width”, “Scale to Page Height”, or “Scale
To Page” from the File menu at the top of the layout editor (fig.19).

If you select “Avoid Page Breaks” from the same menu, FlowJo uses your paper type/shape but places tiles on the
page such that tiles will not cross page boundaries.

Once you have arranged the tiles exactly the way you like, you can print them – you will know exactly how they
will appear on the pages.

Copying and Pasting Graphs from FlowJo

In addition to exporting, you can take an individual graph, or set of graphs out of FlowJo and into a presentation

42
or publishing software like Powerpoint or Word. Do this by selecting the desired graphics, choosing “Copy” from
the Edit menu and then pasting the figure into the destination document.

To access the workspace at this point of the analysis, open the Lesson_6.wsp file.

Lesson 7: Creating Batch Graphical Reports

In this lesson, you will continue with what you learned in Lesson 6 and generate graphical reports for entire
experiments. You will learn how to cycle the layout through different samples in the workspace and how to create
a combined output for printing or exporting of graphs for every sample.

This lesson builds on the workspace you finished from Lesson 6; alternatively, you can open the workspace named
“Lesson_6.wsp”.

One of the strengths of computers is how they handle repetitive tasks. Show the computer how you want something
done once, it can repeat the task a million times without complaint. Flow cytometry often requires application of
the same analysis to any number of samples, and FlowJo’s ability to batch through a set of samples to produce a
graphical report is the heart of FlowJo’s Layout Editor. At the same time, computers are very literal and so it is a
1. Build a Prototype tile
2. Know the type of batch you are looking to perform
3. Double check key “successful batch” iteration controllers
4. Batch the prototype
good idea to know exactly what information FlowJo needs available in order to batch successfully.

In every batch FlowJo will produce a series of tiles, each one containing graphs and statistics and/or other elements
from one or more tubes.

The overall process of producing a batch report is as follows:

To begin examining how to set up a batch in

FlowJo, open the layout editor by clicking on the
layout editor icon in the workspace. When you
drag items into the layout view, FlowJo by default
shows you the desired graph for the sample from
L7 fig.1 - Iterate by sample butttons
which you dragged the subset. FlowJo can also show
1. The sample has the parameters that are displayed in the graph (like Forward Scatter, FL3, FL4, etc.).
2. The sample has a gating tree that has exactly the same subset as what is desired in the graphic (i.e., if the graph
is generated from a CD3 subset of Lymphocytes, then FlowJo looks for a CD3 subset of Lymphocytes in the
current sample).
you the corresponding graph or graphs from any sample in the current group. To do this, return to the layout titled
“B-Cell Plots”, and change the iteration box from “Off ” to “Sample” (fig.1). Choose one of the samples from
the list that appears next to the iteration box once a sample has been selected. The up and down arrows allow
you to scroll from file to file.

43
Batch Plotting

FlowJo will generate a graph for any

given layout item for all samples in
a group during batch processing if L7 fig.2 - Batch settings
the samples meet all of the following
criteria:

At this point you have only looked

at the layout report for individual
samples, one-by-one. To look at
graphs for all samples at once, click on
the “Create Batch Report” button in
the center of the layout editor ribbon.

You can set the output format of the

report, the iteration style, geometry of
the rows and columns and the order
in which plots will be placed as either
going down or across. The set of plots,
graphics, statistics, text and anything
else included in the layout page for one
sample is called a tile. Set the batch to
place the tiles in 1 column as shown in
the image at right above (fig 2) and select
“Down”.

Leave the default output “New

Layout” selected and click “Create
Batch Report”. The result will look like
the figure to the right (fig 2b).

Observe that a new layout page has

been created named “B-Cell plots-
Batch-1”. FlowJo has created the same L7 fig.2B - batched column
set of plots, stats, and text that you
created for sample A1 for the rest of
the samples in the B-cell group. Since
there are 18 samples in the group, there
are now eighteen sets of two plots, a
statistic and a text box.
L7 fig.3 - Batch options
Other Batch Outputs

In the image below (fig 3) you will see that if we return to the original layout and press the “Batch” button again, the
batch layouts can be generated in six different modes, each accompanied with an icon.

The first item listed is “New Layout.” We used this previously to create a new layout in the layout editor, with all
of the iterated samples shown. This is the most flexible way to generate a report, as you can edit the layout further
44
 by adding titles or annotations to specific graphs, or by removing
unnecessary graphs. Unlike other batch exports, batching to a new
layout has the advantage that the resultant layout will update “live”
and reflect changes made in the workspace.

The PowerPoint option produces a pptx file with one slide for every
tile created by the batch option. In the B-cell group we have nine
files, so batching to PowerPoint button will create a PowerPoint file
with 18 slides, each with the two graphs, one statistic and one text
L7fig.3(detail) - Batch options box.

The Printer option allows you to print all of the batched plots.
FlowJo aligns the tiles, ensuring that they are the same size. If you
click on the “Printer” option and
then click “Create Batch Report”,
you will see the dialogue box below
appear (fig 4).

From this dialogue you can select

the paper size, orientation and
how many tiles will appear on each
page by increasing the number of
rows and columns. In the following
figure, three rows and three columns
have been selected. It is important
to remember that when batching
directly to the printer, FlowJo uses
the pages as specified by the page
breaks to determine the tiling. So
L7fig.4 - Batch printer dialogue

you should choose the Scale to Page function in the File tab of the layout editor before selecting this batch option.
One last important piece of information; the Layout Editor view is live in that whenever you change a gate or
analysis, the view is automatically updated to reflect the change. However, Web Reports, any PDF you may generate,
and Movies are NOT live. If you create one of these reports and then change a gate, the report is NOT UPDATED,
because the destination is outside of FlowJo. In order for these media types to reflect changes, you will need to
regenerate the batch output.

45
Try batching to each type of output.

The iteration band contains the iteration controls. This is the same set of options as provided when batching from
the Table Editor. Lesson 8 will discuss in detail how these options apply to layouts.

To access the workspace at this point of the analysis, open the Lesson_7.wsp file.

Lesson 8: Generating Complex Batch Analysis

In this lesson, you will build on what you learned in Lesson 7 to generate graphical reports that iterate the samples
included in a batch in a variety of ways.

This lesson builds on the workspace you have finished from Chapter 7; alternatively, you can open the workspace
named “Lesson_7.wsp”.

Varying Iteration Options

In Chapter 7, you created a layout with

several plots all from the same sample then
batched the layout with the iteration option
set to “Sample”, which forces all graphs to
be derived from the same group during
iteration. To create a batch output, FlowJo
iterates sample by sample through the
current group creating the selected plots
for each sample but for only one sample
per tile.

However, you may want to generate

graphical reports wherein each tile derives
graphics from multiple samples. For
example, you may want to generate one
figure for a panel collected on a particular
day, in which case you could iterate by day.
Alternatively, you may want to overlay plots
from multiple samples for comparison
against each other and repeat that pattern
for all samples. In these cases, you would
like to iterate not over samples, but over
time point or panel. FlowJo gives you this
ability if your FCS data has keywords that
contain such information. This kind of data
L8 fig.1 - Workspace
can be added at the time of collection on
the cytometer or afterwards in FlowJo, as described in Lesson 1.

This tutorial data has a keyword field, “Day”, which lists the experimental time point in days that the blood samples
were drawn. For example, samples A1 and B1 were both collected on Day 0. There is also a keyword field “Panel” that

46
lists which panel of antibodies was
used to stain the PBMC samples.
Right-click on any column header
and select “Edit Columns” on the
right-click menu. This will open
the Edit Column Names dialog
box. Select “Panel” from the dialog
menu, then press the “Add Column”
button, then “OK”.

This will display these two keywords

in the workspace. Now click on the
“All Samples” group to display all of
the files. The workspace will look
like the image above (fig 1).

Create a new layout and call

it “Complex Layout”. Drag the L8 fig.2 - Plots with changes
“Lymphocyte” subpopulation from
sample A1 into the Layout Editor,
and then drag in the “T-cell”
subpopulation from sample B1.
Double-click on the “Lymphocyte”
population and display the graph as L8 fig.3 - Iteration options
a density plot of CD32 versus CD19.
Double-click on the “T-cell” plot and
make it a pseudocolor plot of CD8
versus CD4. To increase the font size,
select the the annotation, then the “Object”
tab. Here you will see the text ribbon and font
size options. Select “18” from the font size
drop-down menu. Your plots will look like
the figure here (Fig.2).

Notice that the batch button is grayed out.

These plots come from two different samples,
so iteration is automatically set to “Off ” and
they cannot be batched without instructing
the software how to iterate. This is because
it is unclear how to proceed with the default
iteration setting of “By Sample” when there are
two different samples to choose from.

At this point we want to tell FlowJo to iterate

by the keyword. From the drop-down box
that currently says “Off ”, select the “Keyword”. L8 fig.4 - Keywords
Then press the key icon and select “Day” (this is what
we will iterate over). The Iteration by Keyword dialog box allows you to choose a value for the starting point and

47
a discriminator. Choose “Panel” in the discriminator then “Okay” to close the dialog box. By selecting “Panel” as
a discriminator you’ve set up the batch to only include files that have the same value for Panel as the sample you
have used to make the original layout. Now from the Value drop-down tab menu, select “0” as the Day starting
point. When you have made these selections, “Batch” will become enabled and the selection boxes will look like
the illustration to the right (Fig.4).

Press the “Create Batch Report” button. The batch

menu will appear as usual, allowing you to select
the output. Accept the default output of “New
Layout”, and change columns setting to “1”. Then
press “Create Batch Report”. You will produce a
new layout that looks like the one below (fig 5).
Notice that you have created a batch of tiles that
contain different samples within them, with each
tile containing samples from the same day, and the
subsequent tiles iterate to the next time point, but
are of the same panel as the picture directly above it.

Batching Overlays with Multiple Samples

L8 fig.5 - Batched plots

L8 fig.6 - Overlayed dot plots

Create a new layout called “Multi-Sample Overlay”. In the
workspace, select the “B-Cell Panel” group, then drag the T-cell population from sample A1 to layout editor.

L8 fig.7 - Iteration settings

48
Double-click on “T-cell” plot in the layout editor and create a dot plot displaying 2H2, a marker for Dengue fever
and CD209, a dendritic cell marker. Then drag the same population from samples A2 and A3 on to that plot,
creating the overlay image below. You will notice that FlowJo automatically changes the format of the graph to
match the existing population. Drag the
legend onto the plot to save space.

Iteration is again in the default “Off ”

setting. This time, select “Panel” from
the iteration drop-down menu and set
the iteration to every “3” samples as
shown in the image to the below.

Now click “Create Batch Report”. Notice

again that the iteration settings match
what was selected in the workspace.
Return to the Multiple Sample Overlay
Layout and this time change the “3”
columns to “2” columns, and press
“Create batch report”. The result is six
plots containing three consecutive
samples, as shown below (fig 8).

Batching While Maintaining

a Control Sample

Assume sample A1 is a control of some

kind that we would like to compare to the
L8 fig.8 - Iteration band
other samples one at a time. To do this,
create a new layout page called “Constant
Control”. Drag sample A1’s “B-cell”
subpopulation into the Layout Editor and
create a Histogram of 2H2. Then drag the
matching population from sample A2
in and overlay it by dropping it directly
on top of the existing plot. Double-click
on the plot and the Graph Definition
window will appear. To set a sample to
remain constant through batching, click
on the “Legend” tab. Double-click on the
“A1.fcs…” item in the box that specifies
which sample should remain locked.
You will note that the text has become
italicized. You can select as many samples
as you wish to remain constant. At the
moment we will select only this one. Now
click “OK”.

Notice that in the legend of the plot, the

sample that will remain fixed is now
L8 fig.9/10 - Final batch

49
listed in italics as well. For emphasis you can use the format box of in the legend to tint the sample that will remain
constant. Your figure will look like the image to the right at this point. Click “Create Batch Report” and set a three-
column layout. The result will be a series of 18 plots; each sample plotted with sample A1, as shown.

In the next Chapter, you will learn some additional Layout Editor techniques, including creating Grids and placing
background graphics underneath the reports.

To access the workspace at this point of the analysis, open the Lesson_8.wsp file.

Lesson 9: Creating Finished Reports

1 This lesson describes the advanced features of FlowJo’s layout editor which are used in creating finished reports.
2 You will learn how to create live text objects containing sample-specific information and statistics, to put in a
3 backdrop containing (for example) logos or specialized forms, and to manipulate Layout Grids – specialized
4 tabular elements that can contain text, tables, graphs, or any other items.
5
This lesson builds on the workspace you finished in
Lesson 8; alternatively, you can open the workspace named
Lesson_8.wsp.

The report that we will generate includes five separate

elements:
an image to use either as a background or a header
a text box containing information regarding the sample L9 Fig.1 - logo in layout editor
a series of graphs demonstrating the gating strategy to achieve a target population
a grid containing the target population for each of nine samples
a table displaying the statistics for all samples

Importing a Graphic
T
Open the layout editor, and create a New
Layout named “Final Report.” From the Edit
Menu, select “Insert Graphic” and choose
an image file of the logo of the institution
where you work. We will include a FlowJo
icon in the top left corner. Alternatively,
you could select a larger image; stretch it
to cover the entire layout editor and drop
plots and text boxes onto it, thus making the
image a background.

Adding Text

Click on the Text Box tool then click-

and-drag a rectangular area that will fit to L9 Fig.2/3- text box options
the right of the image that you inserted. Double-
click in the text box to edit. Right-clicking on the text box opens a drop-down menu with several options. Select

50
“Insert Keyword”. Here you will select several keywords that contain sample-specific information. To insert a
keyword value, click on sample A1.fcs then select “$DATE” (date of sample collection) from the Add Keyword
dialog box. Depress the “Ctrl” button on the keyboard (“Command” on Mac) and select “$CYT” (cytometer used
for collection) as well. Click “OK”. In your own datasets, you may have other keywords pertinent to your research
which you can add using these rich text elements.

If you double-click on either keyword

in the text box, you will note that the
keyword command is bracketed in green
“[…]”. Do not make edits within these
brackets. However, you may add text
between sets of brackets. For example,
you may add explanatory text (and hit
return to create line breaks) to format the
text box, shown in the following image.
From the Object tab, set the Fill Color
(background color) to gray, the text
color (called simply “Color”) to red, and
the Style to bold. When you are finished,
click “OK,” and magnify the view to
100%. The layout editor should look
similar to the figure below right (fig 4).

This rich text and formatting allows

you to highlight your results and add
sample annotation alongside graphical
objects. If you wished to have a
particular keyword value in a different L.9 Fig.4- Text box options applied

font or color, you would have to make

a separate text box for it and format it
accordingly. You can further demarcate
the text from the rest of the layout by
drawing a line using the Line icon, also
in the tools of the layout editor. Double-
click on the line itself to make edits for
color or thickness in the object tab.

Adding Plots

The next task is to add plots to your

layout, showing the gating strategy used
for each antibody panel. This is similar
to the layouts created in previous

L9 fig.5- Annotated plots

51
chapters. First, drag the “T-cell” subpopulation from sample A1 and drop it below the graphic inserted previously.
Double-click on the plot to open the graph definition window and change its attributes to a density plot of CD209
versus 2H2, with outliers displayed. Your graph will look like the plot to the right. Click “Apply”. To display the
gating strategy, click on the “Annotate” tab in the Graph Definition window and click the “Ancestry” check-box.
Click “OK”. You will see two smaller plots appear to the right of the original showing the gates used to get to the
target subpopulation.

L9 fig.6 - Annotated plots with B-cells

Next, drag in the “B-Cell” subpopulation

from the same sample (A1) and place it
next to the “T-Cell” plot. Double-click
on this plot and change it to a Contour
plot of CD32 versus CD19 and click
the check-box for “Backgating.” Click
“OK.” In the layout editor two plots
will appear again, as shown below. All
events will be colored grey with any
cells that would be included in the
downstream gate (regardless of whether
they were gated out in an earlier step)
are back propagated onto the preceding

L9 fig.7 - Grid tool

52
plot and colored red. This is a tool to assess the effect of a gating strategy, as gates may restrict what you analyze in
a subpopulation.

Using the Grid Tool

Now we will add a grid to your layout. Choose the grid tool from the toolbar above your layout in the Layout Editor.
Click, drag, and release within the layout editor to draw a grid. Right-click, choose cell dimensions to set the number
of rows and columns to 3 rows and 3 columns. You can now drag populations directly into the grid box and they will
snap to the size of the box. You can also drag text boxes or statistics in to create tables or annotation.

Create a text box, and type in it a sentence or two stating that the grid will hold the B-cell population for
samples A1 – A9. Then, drag and drop that text box into the upper left grid box. Next, drag and drop the B-cell
population from each of those files into the grid. A quick way to do this is to select the “B-cell” population in the
workspace for sample A1 from the “B-Cell Panel”
group, then open the “Edit” menu and click on
“Select Equivalent Nodes”. You will notice that this
will highlight all of the B-cell populations. Click

on a “B-Cell” population
and drag-and-drop it into
the upper right grid box.
The selected populations
will fill out the rest of the

L9 fig.8/9 - Final layout with table

grid boxes.

Adding a Table from the Table Editor to the Layout Editor

53
Finally, we will add a table of statistics. We can reopen the Table Editor by clicking on the table editor shortcut
button from the workspace. Then choose the “B-Cell Table” and select the batch to current layout icon. The table
will appear in the currently open layout page, which is the Final Report page. You can scale the table by grabbing
a corner and dragging it larger or smaller. Your layout should now look like the following figure.

*You may need to adjust the paper size in the print dialogue box to allow for your table to fit your layout.

From here you can print a report, publish to the web, or generate a movie or pdf. These options are explained at
the end of lesson 6.

To access the workspace at this point of the analysis, open the Lesson_9.wsp file.

Lesson 10: Compensation

Lesson 10 does not build on the previous lessons. Rather it is an independent tutorial on the compensation tools
within FlowJo, using the data set that you have become familiar with through the first nine lessons.

This tutorial also does not serve as a comprehensive guide to what compensation is and why we do it. There are
many good references that deal exclusively with the subject. A tutorial that we recommend is:

M. Roederer. Compensation: An informal perspective. May 2000. Web 18 April 2013. www.drmr.com/compensation

For the purpose of this tutorial it will suffice to explain that when more than one fluorescent probe is used in a
cytometric experiment, the emitted fluorescence spectral outputs overlap, adding a “noise” component to the
measured intensity of all parameters. The contribution of the noise component can be determined by collecting
tubes containing cells stained with a single fluorescent color. These single stain controls allow us to measure the
intensity produced across all colors that is due to the single color used in that control tube. All cells produce some
amount of autofluorescence so we do not assume that median fluorescent intensity (MFI) for a single stain control
in channels other than its collection channel should be zero. Rather, we assume that the MFI of these parameters
should be equal to that of the background.

Compensation is the process of correcting the spectral overlap that occurs during multicolor flow cytometry. This
is done by quantifying each dye with which a particular cell is labeled, and subtracting the amount of intensity that
adjusts MFI of single stained cells for all parameters they were not stained with to the background MFI.

Compensation is applied in one of three ways:

1. Using an algorithm on the acquisition software before
FCS files exported and loaded into FlowJo
2. Post-acquisition in FlowJo using an algorithm
3. Manually on the cytometer

In the last case, the record of the compensation performed,

the matrix created, and the raw (non-compensated)
L10 fig.1 - Add comp matrix column

54
data are not stored in the FCS file. (Most software compensation algorithms will produce very similar results,

making algorithmic L10 fig.2 - Compensation matrix added compensation more robust than
manual.) Therefore, you will not be able to edit the matrix or examine uncompensated parameters, though

L10 fig.3 - Matrix editor

the data can be analyzed. In this tutorial, we will thus focus on the first two cases.

Data Compensated by the Acquisition Software

Load in the folder Exp. 1, Panel_1.1. The grid displayed next to each of these samples indicates that there is a
compensation matrix associated with these samples. There are two ways to see which matrix is associated with
which sample. First, go to the workspace and right-click on a column header. Click “Edit Columns” and select the

55
keyword “Compensation Matrix”, then “Add Column”. Click “OK”. (fig 1)

The workspace will look like the figure below, showing that all files were compensated with the Acquisition-defined matrix.

Alternatively, you can double-click the grid to open the Matrix Editor. Click on the matrix next to sample A1.

L10 fig.4 - New matrix

The box on the top shows the compensation matrix (fig 3). If you look at a box in the grid, denoting the intersection of
two parameters, the grid can be read as “X.XX %” of the measured intensity of the row parameter is being subtracted

L10 fig.5 - Matrix renamed

56
from the column parameter. So in the figure above, for example, 0.7384% of the PE-A intensity is being subtracted
from the FITC-Alexa intensity. The bottom panel shows Histograms of compensated data for the selected population
in the selected sample, and allows one to compare this to uncompensated data. The left panel shows the matrices
that are available in the current workspace and allows you to edit their color formatting and name.

Editing an Existing Matrix

FlowJo will not allow you to modify your raw data files, nor the compensation matrix. However, if you would like
to modify your compensation matrix in this FlowJo workspace, click “Edit” at the top of the matrix, a confirmation
window will pop up, click ok and then you can click in any cell of the matrix to edit it, type in the new number, and
then press the “Enter” key. By default, editing a matrix creates a new matrix, visible in the matrix navigator (fig 4).

Rename the edited matrix from “Acquisition-defined-copy” to “Matrix2” by clicking on the text in the left panel
and choose purple for its color formatting (fig 5).

By default, you will see that this matrix has been applied to the Panel_1.1 group. To change the compensation matrix
that is applied to a sample or a group, simply drag-and drop the [M] icon onto either a sample in the bottom part
of the workspace or a group
in the group analysis pane,
respectively. Finally, right-
clicking on the matrix badge
in the workspace allows you
to edit the matrix, to re-apply
the acquisition matrix, or re-
apply the acquisition matrix
to the group.

A word of warning - we
discourage random matrix
editing. The algorithmically
calculated values are the
correct values for the
compensation controls that
were presented. However, it
is possible that inappropriate
controls were used and
correction is needed. The
best way to accomplish
this is to find a population
“positive” for a single
parameter and gate on this
positive population. Then
modify the compensation
matrix and check that there
is a nearly equal MFI of
background
L10 fig.6/7 - Group definition for matrix editor
and MFI of the parameters for which

57
you expect the population to be negative.

Creating a New Matrix

If you have single stain control sample

data, FlowJo has the ability to calculate
a new compensation matrix, even if the
data files were compensated previously by
the acquisition software. In order to create
a new matrix in FlowJo with single stain
controls, load the compensation controls
folder into the workspace. Once loaded the
workspace will look like the figure below.

You will note that the pink Compensation

group has populated with 7 single stain
sample files - there is a single stain control
for every color except 7-AAD, which this
experimenter chose not to compensate.
These samples have been automatically
loaded into the compensation group, based
on its group definition (fig 6/7). To examine
this group definition, double-click on the
compensation group, and you will note that
this group has been programmed to look
for file names ($FIL) that contain the text
“comp” or “unstained.” L10 fig.8 - Single stain controls in compensation editor

L10 fig.9/10 - Adjusting controls

L10 fig.11 - Adjusting controls

58
 Close this window, and we will
create a new matrix using the
Compensation Editor.

With the compensation group

selected, the compensation editor
icon will become live - press this to
open the editor.

FlowJo will automatically attempt

to match the controls samples to
the proper parameter using peak
finding - that is, FlowJo scans the
data and attempts to identify which
tube has a “positive” (fluorescence
above background intensity)
population for a parameter. Since
these are single stain controls, there
should only be one parameter per
file with a positive peak. (fig 8)

Regardless, we recommend that

L10 fig.12 - Compensation control

you check the file names against

the parameters to make sure
FlowJo has done the assignment
properly. In this case, three

L10 fig.13 - Compensation complete

59
adjustments must be made so that the matrix calculation can be finalized:

Since no compensation control was acquired for 7AAD, click on the sample drop-down menu next to 7AAD and
select “Remove This Parameter”. For APC_Ax 647-A (2H2), select the proper single stain control using the sample
drop-down menu to “CompCtrls AF647.fcs”. (fig 9/10)

Correct the sample selection for APC-

Ax700-A (CD4) to “CompCtrls AF700.
fcs”. FlowJo will automatically set gates
for positive and negative populations on
this sample. (fig 11)
The compensation editor should now
look like the screenshot below (fig 12):

A compensation matrix has now been

calculated, and the status in the upper
right-hand corner of the compensation
editor should now read “Finalized.” In
order to apply this matrix, you may
either drag and drop the [M] button
onto a sample or group, or alternatively,
use the “Apply to Group” drop-down
menu at the top left-hand corner of the
compensation editor. Go ahead and
apply this new matrix to the Panel_1.1
group, and you will observe that the
compensation badges next to the samples
L11 Fig.1 - preferences dialogue
in this group indicate that the FlowJo-
calculated matrix has been applied to
these samples (compare the matrix color
under the Compensation group with the
badges next to the samples). (fig 13)

This concludes the compensation

tutorial. This work is saved as Lesson_10.
wsp.

Lesson 11: Setting the

Preferences
Lesson 10 is an easy finish; there is no
work to do here. This chapter is simply

L11 Fig.2 - workspace preference

60
aimed at highlighting some of the more commonly used preference settings in FlowJo to help you start customizing
the software to your needs and to emphasize that most aspects of FlowJo are customizable.

What are preferences?

L11 Fig.3 - graph preferences

Preferences are the customizable set of defaults

that FlowJo uses throughout the program. If you
find yourself repeating an action over and over L11 Fig.4 - font preferences

again, consider that you might be able to set the program to do what you like by default. The preferences are
accessed through the Edit menu. When you click on the
preferences icon, the interface shown to the right will be
displayed. (fig 1)

Each icon controls the settings for the listed aspect of

L11 Fig.5 - gate preferences

FlowJo.

The workspace menu (fig 2) allows you to control the

workspace appearance. There are controls for borders,
decimal precision, file sort order, and of special L11 Fig.6 - layout preferences

interest, the choice of which keyword is displayed

under the “Name” column in the workspace. That

61
interface is shown below.

FlowJo does not allow you to directly double-click and rename a file in the workspace, but from this interface you
can control what appears by selecting the keyword or combination of keywords that contains the information for
identification of each file. If there are no existing keywords that display the information you need, you can create
your own keyword and then choose it from this menu.

The Graphs menu (fig 3) allows you to control the default appearance of graphs. The type of graph and its options
can be set using the interface below.

The Fonts menu (fig 4) allows you to set the font

characteristics for nine different places in FlowJo, or to
unify them.

L11 fig.7 - Preferences annotation

The Gates menu allows you to control the appearance

of gates, the annotations that appear on screen when
a new gate is created, and what to display when you
double-click on a gated population. The entire menu
L11 fig.8 - Preferences file types
is shown in the figure below at left (fig 5).

The Layouts menu (fig 6) allows you to control what happens in many aspects of the Layout Editor. One of the most
important preference in this menu is the “Drag Mismatch Policy” drop-down menu settings.

Drag mismatch tells FlowJo what to do when you drag plots from two different samples into the layout editor. “Turn
Off Iteration ” will allow you to place any plot into the layout. Batching will then become disabled, as FlowJo will not
know what sample to iterate. Therefore if you want to batch, you will have to specify a keyword by which to iterate
and discriminate (see Lesson 8 for details).
“Coerce to Current Iteration” forces all of the plots to come from one sample, so FlowJo will change what you drag
in. “Reset Iteration Value” will change the iteration value to match the selected samples, if this is possible. “Ask” will
cause FlowJo to bring up a pop up window asking you to specify what you would like done every time you drag
different samples into one layout.

The Annotation menu allows you to specify which keywords are automatically displayed when a sample is dragged
into the layout editor. It also allows you to define the color order for overlays and define what special features
appear on plots by default. The main portion of this menu is shown below left (fig 7).

The File Formats menu (fig 8) allows you to specify what the default file output will be for exporting from the graph
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