Flowjo

Data Analysis Software for Flow Cytometry

Version X User Documentation

Windows/Mac user documentation

Basic Tutorial
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 web site are copyright © Tree Star, Inc. 1997-2013. All rights reserved.

• Basic Tutorial •
• © MMXIII •

Revision date: 16 April 2013

version 10.0.6

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 Table of Contents

Introduction............................................................................................iii
Getting Started.......................................................................................1
Lesson 1 - The Workspace..................................................................3
Lesson 2 - Gating...................................................................................7
Lesson 3 - Statistics...............................................................................10
Lesson 4 - Batching in the Workspace...........................................12
Lesson 5 - Basic Table Editor Functionality...............................14
Lesson 6 - Basic Layout Editor Functionality............................18
Saving.........................................................................................................25

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Introduction - FlowJo Basic Tutorial
FlowJo is an integrated environment for viewing and analyzing flow cytometric data, presented in the form of a
Workspace. The Workspace contains a list of all of the data samples that you load, the gates, statistics and other
analyses that you apply, and the table and graphical layout that you design. The Workspace is saved as a FlowJo
document on your hard disk; when you reopen the document, you will see the status of your analysis as it was
when it was last saved.

This tutorial is designed to introduce you to the basics of 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.

This tutorial is designed as an introduction to FlowJo. The 8-color PBMC Advanced Tutorial provides more detail
on functionality. Furthermore, FlowJo is capable of much more that simply can’t be covered in an introduction
such as this (for example, there are analysis platforms to perform DNA/Cell Cycle analysis, Kinetics analysis,
Proliferation and Population Comparison). You can learn more about FlowJo through the online help tool.
Whenever you ask for help from FlowJo by pressing the question mark button in the interface of any window, it
launches a web browser and accesses help pages about the part of the program you are using. You can navigate
through the help pages to find out more about all of the aspects of FlowJo. In addition, FlowJo.com contains a
page of FAQ’s (Frequently Asked Questions), tutorial videos on many FlowJo topics, and the Daily Dongle, a
searchable 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 always download the most recent version of FlowJo
from: http://flowjo.com/download/index.html

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Getting Started
To begin this tutorial you will first need to install FlowJo. The easiest way to do this is to download the installer
from our website: http://www.FlowJo.com/download/index.html

You can 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 on the icon to launch FlowJo.

You can download the tutorial and Sample Data here : http://flowjo.com/home/tutorials

You have downloaded the full version of FlowJo, however, until a serial number is provided, FlowJo will only load
specially enabled demonstration data files, such as those provided for this tutorial. To obtain a trial 30-day serial
number so that you can try FlowJo using your own data simply fill out the form at:
http://www.FlowJo.com/FLS/registration/trial.html

When you launch FlowJo for the first time, the FlowJo license information window will pop up. In the welcome
tab (see screenshot below), you must agree to the license terms and click next.

This takes you to the institution tab where you can

select and input the type of license you have. For the
purposes of performing this tutorial, you need only
to select “Continue under (free) student license”
and click next.

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If you have an individual license that you would like to use, you can input your serial number in the window
(below left). 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. If you have an enterprise group license,
you can configure the connection here as well (below right).
For more information about licenses, visit www.flowjo.com/home/licenseoptions.html

Click here to use a serial number

The next tab is “Cytometry” (below left), which allows you to fill out a little information about the type of
experiments and analysis you do so FlowJo can automatically tune your default settings. For the purpose of this
tutorial, you can leave this as is for now and click next.

The last tab titled “You” asks a little more about your FlowJo usage. Make sure ‘Experienced’ is selected under
FlowJo Knowledge section so you will have access to all options in the tutorial. Click done. FlowJo has now
successfully launched and you can begin the tutorial.

The data and accompanying workspaces can be acquired from: http://www.flowjo.com/home/tutorials/

Following this link will bring you to a page where you can download the Basic Tutorial zip file 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 it on a Mac and right click to extract them on a PC.

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Lesson 1 - The Workspace
The data we are using for this tutorial are from a basic antibody titration experiment. There are five samples with
varying dilutions of PerCP-Cy5.5 anti-CD8 antibody from 1:100 to 1:1600. When you open FlowJo, you will first
be presented with the Workspace, which is your starting point in FlowJo. The ribbons contain tabbed bands, such
as the FlowJo band above, and scrolling through the various bands will display the different tasks in each band.
Each band can be customized with your preferred tasks which is a great way to customize any band with features
you most commonly use.

This is a great dataset, not only to explore the basic functionality of FlowJo, but to also begin to understand how
to properly titrate antibodies (a necessity for ensuring proper results and preventing waste).

The workspace is your starting point in FlowJo. There are

three major components:

1) The Ribbons, which organize the tasks in FlowJo at

the top of the interface:

Lesson 1 Fig.2 - ribbons

To remove a task, expand the band to full view, grab it using

its text label and drag it out of the ribbon.
Lesson 1 Fig.1 - workspace

Lesson 1 Fig.3 - bands

To add a band, click on the Ribbon Configuration icon and simply drag the
icon to your ribbon.

Lesson 1 Fig.4 - ribbons menu

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2) The Group section:

Groups in FlowJo allow you to

organize samples for common
analyses. For example, you may want
to apply an analysis strategy (batch) Lesson 1 Fig.5 - groups
to a group of commonly stained
samples. To learn more about groups,
please see the 8 color PBMC Tutorial.
In this tutorial, we will not use groups
for the sake of simplicity. We will
discuss batching in future lessons of
this tutorial.

3) The Sample section:

Lesson 1 Fig.6 - adding samples to workspace
This section will contain a list of your samples.

In fact, let’s go ahead and load the samples.

Go to the Basic Tutorial folder and drag the
files into the workspace:

Your workspace should now look like this:

You’ll notice the files are named with the default

file name keyword ($FIL), which was written
by the cytometer to the FCS sample file.

Keywords are “metadata” contained within

the file, in addition to the fluorescence values
of every event. Keywords are a great way to
annotate data during acquisition. (A list of
commonly used keywords and use cases can be
found in your workspace preferences.) Many
Lesson 1 Fig.7 - samples in the workspace
of these keywords are recorded during data
collection and every aquisition program allows
you to add additional keyword values. Keywords
can also be added in the FlowJo workspace.

Keyword naming convention can be changed

in the workspace preferences if you prefer to
have a different keyword used as your naming
convention. To explore the keywords of your
files, right click on a file and click Inspect.

Lesson 1 Fig.8 - sample right click options

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You can explore the keyword values of your file in this interface and we are particularly interested in a specific
keyword we entered during acquisition of these data called ‘TUBE NAME’.

Lesson 1 Fig.9 - sample inspection window

This keyword was used to denote the dilution of antibody used in each tube. Click OK to close this window.
Annotating your .fcs files during acquisition with keywords, or metadata, is a great way to quickly add important
information to your analysis. We will explore these various ways to use the metadata in subsequent lessons.

Let’s add this TUBE NAME keyword to our workspace so we can quickly reference the dilution used in each sample.

To do this, go to the Configure Tab and click ‘Edit Columns’:

Lesson 1 Fig.10 - configure

Now, add the TUBE NAME keyword to the
workspace. We can now see the dilution value
keyword for each sample in our workspace:

Lesson 1 Fig.11 - column edit dialog

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Because FlowJo does not know
a ratio formula as a keyword, we
need to add a keyword for the
antibody concentration. Click on
the Workspace tab and select the
Keywords band, click the button to Lesson 1 Fig.12 - tube name column
Add Keyword.

In the Add Keyword dialog box,

type “Antibody Concentration”
and click ok. Another column is
added to the workspace just like
when we added the Tube Name
keyword. Double click in the first
cell and type just the dilution
amount. For Sample A01, this
number is 100. Do this for all
samples in the workspace. When
finished, your workspace will look
like the one below.
Lesson 1 Fig.13 - add keyword

Lesson 1 Fig.14 - antibody concentration column

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Lesson 2 - Gating
You can start this lesson (skipping lesson 1) by opening the Lesson 2.acs file.

Gating is the process of subsetting collected events for further analysis. Subsets can be gated to generate further
subsets, until a collection has only the cells for which a graphic display or analysed statistics are desired. FlowJo
offers a whole set of gating and advanced gating tools to facilitate this process.

To begin creating gates, we must first open the graph window for the first sample. To do this, double click on the
sample A01. This will open the graph window:

Gating options are in the top left hand corner. We are interested
in examining lymphocytes, so lets create a polygon gate on the
lymphocytes. Select the polygon gating tool:

Click the mouse to make a gate node and then continue

clicking to draw a gate around the lymphocytes. Double click
to close the gate. FlowJo will then ask you to name the gate.

Lesson 2 Fig.1 - graph window

It may suggest the correct gate name, in this case Lymphocytes.
You can leave this gate name or enter another name. We’ll leave it
as “Lymphocytes”. If you click the “+” button in this dialog, your
gate name will be saved for use at another time.

Double click within the lymphocytes gate in the Graph Window

to isolate just the lymphocytes.
Lesson 2 Fig.2 - gate naming
Notice how the gated population is created in the workspace as well below and indented from the total sample.
This is FlowJo’s revolutionary hierarchical gating which immediately indicates the gating strategy and derivation
to the user.

Lesson 2 Fig.3 - sample analysis heirarchy

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Change x and y axis parameters to match the picture below on the right (fig.4).

You’ll notice that we have isolated

the lymphocytes. Since we are
doing a titration on CD8a, we
will now change the x-axis of the
lymphocyte population to CD8a
and the y-axis to histogram. Do
this by clicking on the currently
displayed parameter label. It will
drop down a list of all additional
parameters:

Lesson 2 Fig.4 - lymphocyte gate

Lesson 2 Fig.5 - x-axis

Lesson 2 Fig.6 - y-axis
There are a couple of things to note about data display in the Graph Window:

*FlowJo applies a biexponential transformation automatically to digital (fcs3.0) data. There is a preference to turn this off, but
it is actually a preferable way to display digital data. The T button also allows you to customize the axis to adjust the transform
or convert the axis to log or linear.

*The parameters say comp-parameter name because these are digital data (fcs3.0) files that were acquired with a compensation
matrix. FlowJo applies the compensation matrix generated by acquisition software automatically upon loading data. If you
did not compensate on the machine or use fcs2.0 data, the parameters will not have the comp-prefix. Compensation is
described in more detail in the 8 color PBMC tutorial.

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To set a gate on the negative and positive populations, let’s select the bisector gate tool and draw a range gate on the
negative and positive populations. FlowJo will name each gate accordingly.

Lesson 2 Fig.7 - samples

*Typically we would gate on a control (unstained, Fluorescence
Minus One or Biological control) to properly set gate boundaries.
However, with a titration experiment, this isn’t always necessary.
Therefore, please consider this an exercise in gate creation and
not in theory on how to properly set gate boundaries.

This concludes Lesson 2. You can always find more information

on gating and gating options at flowjo.com.

Lesson 2 Fig.8 - bisector gating tool

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Lesson 3 - Statistics
You can start this lesson (skipping lessons 1 and 2) by opening the Lesson 3.acs file.

In version 10 of FlowJo, there is now a ‘quick statistic’ option to calculate the median, CV and Freq. of from the
Statistics band. Since flow cytometry data is typically NOT normally distributed, we refer to the Median as a MFI.

Lesson 3 Fig.1 - adding statistics

To add the MFI (Median Fluorescence Intensity), CV (Coefficient of Variation) or Freq. of … (Frequency of a
population), these ‘quick statistic’ options can be used.

MFI technically stands for Mean Fluorescence Intensity, but a mean assumes a normal distribution. Since flow
cytometry data is typically NOT normally distributed, in flow cytometry, we refer to the Median as a MFI.

In this experiment, we want to know the CD8a MFI, or CD8a expression level, of the CD8a+ and CD8a-
populations. Therefore, all you need to do is select the CD8a+ population in your workspace.

The MFI is a measure of the expression profile of a particular parameter. Typically, MFI is used when looking at up- or
down-regulation of a marker, or fluorophore expression like GFP. In this experiment, we use the MFI to calculate the
separation/staining/sensitivity index. There are many formulas to calculate these different indices, but a very simple
way to do it is to take the MFI of the positive and divide it by the MFI of the negative. For titration experiments, this
basic formula is sufficient and will be described in more detail in a later lesson.

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We will now go to the quick statistic interface and choose Median>Comp-PerCP-Cy5.5:: CD8a

Lesson 3 Fig.3 - median

This will add the MFI (CD8a expression
level) to your CD8a+ population.

Now, do the same thing to the CD8a-population. Lesson 3 Fig.4 - median statistic applied to CD8a+ population
You can also drag and drop the MFI for CD8a
from the CD8a+ to the CD8a- too.

Additional statistics are available through the

‘Add Statistic...’ interface such as Mean, Standard
Deviation (SD), Percentile, and Count. Lesson 3 Fig.5 - median statistic applied to CD8a- population

Count is a bit redundant since it is in the workspace already, but if looking to export the count of population(s) out of
FlowJo, adding the statistic to the workspace allows for drag and drop to the Table or Layout Editor.

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Lesson 4 - Batching in the Workspace
To skip to lesson 4, open the Lesson 4.acs file.

Batching is a process that performs an operation automatically for a group of files, rather than having to manipulate
one file (or sample) at a time. Almost everything in FlowJo can be batched because batching saves you time! As
FlowJo is developed by leaders in the field, any feature to save time has likely been implemented. In this Lesson,
we will discuss batching in the workspace before we discuss batching to outputs using the Table Editor (Lesson 5)
and Layout Editor (Lesson 6). In Lesson 4, we’ll discuss batching in the workspace.

A single gate, a single statistic or an entire gating tree can be batched. In this experiment, we want to batch our
entire tree to the group so that the gates and statistics we added to sample 1 are present in every sample. To do this,
select the Lymphocytes population in the workspace, hold down shift on your keyboard and select the bottom
statistic. You should have the entire gating tree highlighted now:

You can drag and drop this to any

group. Go ahead and drag this
gating tree to the All Samples group.

What you’ll notice is that the

gates and statistics have now been
applied to every sample in the Lesson 4 Fig.1 - selected analysis
group. The initial gate location for
every sample is based on Sample 1.
FlowJo allows you to go through
each sample and adjust the gates on
a per sample basis. FlowJo will not
propagate a change made in any
one sample to all samples unless
you drag the altered gate to the
group. This allows you to control
each sample’s gates and choose
when you want to re-batch any
gate to the whole set of samples.

On the other hand, if you change

a gate in one sample and want it
to revert to the original group
applied gate, just select it in
the workspace and click delete
(backspace) on the keyboard.
The altered gate will reset to the
group-owned gate.

Lesson 4 Fig.2 - selection applied to all samples

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Use the arrow keys in the graph window to scroll through plots
and check gates.

Alternately, with two or more graph windows open, you can hold
down the shift key on the PC or the control key on the Mac and
scroll through more than one Graph Window at once. This allows
you to check multiple gates simultaneously.

Any gate that is modified will turn black and unbold. Group
owned gates/statistics receive the formatting of the group to which
they have been applied. Here you’ll notice all gates/statistics are
black and bold because the ‘All Samples’ group is black and bold.
If the group was a different color, the group-applied gates/statistics
would inherit that different color. Modifying gates after they have
been applied to a group is acceptable in many circumstances
depending on your analysis. FlowJo notifies you of the change by
turning the population black and unbold.
Lesson 4 Fig.3 - graph window navigation

As stated above, select a modified gate and

click delete on the keyboard to have it reset
to the group-applied gate or drag it to the
group to apply the altered gate to every
sample in the group.

Lesson 4 Fig.4 - group applied analysis

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Lesson 5 - Basic Table Editor Functionality
To skip to lesson 5, open the Lesson 5.acs file.

The Table Editor in FlowJo allows you to quickly create tables for export into a spreadsheet program like Excel
or to a database. The Table Editor is solely for creating statistic tables and does not manage graphics. Exporting
graphics will be discussed in Lesson 6.

To open the Table Editor, click on the

grid icon button in the toolbar
or the Table Editor button in the
FlowJo band.

When you open the Table Editor,

the first interface is blank. Here, we Lesson 5 Fig.1 - table editor button locations
will drag both Median statistics from
Sample 1 to the Table Editor.

Lesson 5 Fig.2 - adding statistics

Statistics only have to be placed into the Table Editor
from one sample in the group. When the table is
created, it will batch automatically for all samples
in the group.

We now have our CD8a MFI, or expression intensity,

for the CD8a+ population and CD8a- population in
our Table Editor. To change the name of the column
header, just click in the rightmost cell for each
statistic. Add custom names for both statistics as
depicted in the figure below (fig.3).

Lesson 5 Fig.3 - custom named columns

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Next, we want to add a keyword to the table, click on the Edit tab then click on the Add Column button. Next, the
Column Information dialog window will appear. Here you will select the Keyword Tab, and scroll down until you
find the Antibody Concentration Keyword from the column on the left. Now type Antibody Concentration in
the column heading field and click OK. This adds a keyword to your table so you have the concentration for each
sample upon batching and creating a line plot.

Lesson 5 Fig.4 - adding a column

Your table should look like this:

Lesson 5 Fig.5 - new column

We’ve added statistics and keywords. Now, we can also add equations in the Table Editor as well. Calculations can
be done in Excel upon export but you can also do them in the Table Editor before batching.

To create a formula in the Table Editor,

go to the Edit tab and click Add Column.

In the resulting interface, click on the

‘Formula’ tab:
Lesson 5 Fig.6 - add column
Now, for a titration experiment, we want to
calculate the ratio of the expression between
positive and negative. Therefore, we will
create a ratio of CD8a+ MFI / CD8a- MFI,
with a column heading name “Ratio MFI”.

Enter the Column Heading and then select

the Reference CD8a+ MFI.

Now, insert the divide function and insert

the reference for CD8a- MFI.

Lesson 5 Fig.7 - column information dialog

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Your formula should look like this:

Click OK and you will see the formula in the Table Editor.

Lesson 5 Fig.8 - formula

Lesson 5 Fig.9 - table editor

You have set up the Table Editor with statistics, keywords and a formula. Many users only place statistics in the
Table Editor, but using keywords and formulas can save you work down the line.

You can batch now to generate the table for every sample in your group or you can explore the data a bit more.

To set your batch output, go to the Table Editor tab. You can batch to clipboard, to printer, directly to the Layout
Editor, or to a file. The option most users prefer is to batch to FlowJo first. Batching to FlowJo first will allow you to
check the table over before export and then, from the created table, you have all the options of where to export the
table. So, there is no disadvantage to batching to FlowJo first.

Click the orange sprocket icon in the upper right hand corner to start the batch:

Lesson 5 Fig.10 - batch

Here is the resulting table:

Lesson 5 Fig.11 - batched table

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You may choose to “export” or “save” your table data to Excel and write and apply your formulas there. That is fine,
but hopefully you now understand how to do it in FlowJo as well!

Lesson 5 Fig.12 - output menu

To find the saturation point in FlowJo, plot the concentration versus the ratio. Select the Antibody Concentration
row in the Table Editor, hold down the shift key and also select the Ratio MFI row, so both are now highlighted.
Click on the Plots tab in the Table Editor and select Line Plot.

Here we find that the proper dilution

for this antibody is 1:200 because
this gives us the greatest difference
(separation) between positive and
background staining.

Click File > Save As... to choose the

format to save the file as an image file.

Lesson 5 Fig.13 - line plot button

Lesson 5 Fig.14 - line plot graph

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Lesson 6 - Basic Layout Editor Functionality
To skip to Lesson 6, open the Lesson 6.acs file.

The Layout Editor in FlowJo is one of the most feature rich areas in the program. In version 10, it has much of the
functionality that any graphics program has, such as rich text, shading, opacity, rotation, gradient fill, and various
shapes.

In this lesson, we will only cover:

1) Making a report, editing it and creating a simple batch.
2) Overlaying.

The 8 color PBMC tutorial goes into further detail on the Layout Editor and there is a Batching and Iteration
Tutorial available for advanced batching in the Layout Editor.

For more details on the Layout Editor, please visit: http://docs.flowjo.com/vx/graphical-reports

To make a report in the Layout Editor, you need only to create a report for one sample and then when you batch
it, the same layout of the initial report will be generated for every sample in the group.

To begin making a report for our first sample, open the Layout Editor and drag the Lymphocytes gate from the
first sample from the workspace to the Layout Editor.

When you drag a population to the Layout Editor, you will get a graph:

Lesson 6 Fig.1 - layout editor

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When you drag a statistic to the Layout Editor, you will get a text box of that statistic. Drag the Median CD8a
statistic from the CD8a+ population of sample A01 to the Layout Editor Workspace.

Lesson 6 Fig.2 - stats in layout editor

To edit the graph, simply double click on it. The resulting graph
definition window will allow you to edit any of the properties of the
graph in the Layout Editor.

For example, lets edit the x-axis. Go to the Annotate tab of the ‘Graph
Definition’ window and under the ‘X Axis’ section, enter CD8a as the Label.

Lesson 6 Fig.3 - graph definition window Lesson 6 Fig.4/5 - editing layout

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Uncheck show frequencies, then click Apply and you will observe that the label and annotation change:

You can also adjust the font size or style.

*If you find that you have to adjust the

font size/style often, simply go to the
Fonts section of Preferences where there
is a font preference set for axis labels in
the Layout Editor. Changing it here will
cause all new plots in any subsequent
layout to automatically receive the
preference set attributes.

Also, set the color to blue by going to

the Specify Tab and selecting blue as the
Foreground Color.
Lesson 6 Fig.6/7graph definition layout options
Click ok, notice that the histogram lines change color in the Layout Editor. In
the Layout Editor, right click on the histogram. At the bottom of this drop-
down menu, there are options for line style, weight and coloring. Here, you are
going to make the histogram solid by selecting Coloring and the Filled option.

Now, to finish this report, place the CD8a- MFI statistic in the Layout Editor as
well and create a ratio formula right in the Layout Editor.

To make the ratio, create a new

text box and double click to put
the text box in edit mode and
enter in “Ratio of pos/neg MFI:”.

Now, double click the CD8a+

MFI text box and copy/paste the
11184 string from the CD8a+
text box to the new text box. Lesson 6 Fig.8 - color options

Do the same for the CD8a- 43.9

string. Separate them with a divided by or /.
Lesson 6 Fig.9 - fill menu

It is important to copy/paste the string

as this is the link to the statistic. Simply
entering in 11184/43.9 will create the
formula properly for this sample, but when
batching, will simply batch 11184/43.9 for
every sample. We will want to actually
batch the ratio, so we want the statistic
string that you get with copy/paste.

Lesson 6 Fig.10 - resulting layout

20
To create the equation, highlight the text, right click and select “Equation from Selection”.

Lesson 6 Fig.12/13 - menu

The text box should now convert to this:

Lesson 6 Fig.11 - equation

Then change the line style formatting to bold and text formatting
size to 18 point in the Layout Editor to get this:
*Text within a text box must be highlighted for changes to take effect.

One last thing we may want to do is to get rid of the sample annotation.
To do this, you can simply select it and hit delete.

Lesson 6 Fig.14 - ready to batch

Lesson 6 Fig.15/16/17 - layout

You are able to reorganize the layout to how you prefer it and then we’ll batch!

1) Return to the Layout Editor Tab to batch these graphs and statistics, you can select any group you want, but in this
experiment, you want group All Samples.

2) The iterator is off, so it will batch by sample by default. This is what we want. It could also be set to sample with
the same effect. Advanced iteration can be learned in the Batching and Iteration tutorial.

3) We will batch to a new layout, but many other options are available.

4) Set batch orientation to 3 columns going down the Layout Editor.

5) Click the sprocket icon to generate the batch report!

FlowJo will generate the batch report for all the samples in the selected group. Arrange them on five pages so you can see
the results in this document (Obviously, you can also see the results on your screen).

21
As you can see, everything batches. Graphs, statistics, text boxes or other objects will all batch. Batching can save you
an extraordinary amount of time. By setting up the prototypical report for your first sample, you can quickly generate
a report and get every object present for ALL samples by batching.

Overlays

This section can be started by using the

Lesson 6b.acs file.

The Layout Editor allows any sample

or population to be overlayed on any
existing graph of a sample or population. Lesson 6 Fig.18 - batched layout

Creating an overlay is simple. First, add a new layout by clicking the plus
button in the upper left hand corner of the Layout Editor Band.

Lesson 6 Fig.19 - new layout

This can also be done in the Table

Editor to create multiple tables of
statistics. There is no limit to the
amount of layouts or tables you can
make in FlowJo.

In this experiment, we will drag the

lymphocyte population from Sample
A01 into the Layout Editor. Lesson 6 Fig.20 - new batch overlay

(We have removed the frequency, as we did in the first batch):

22
Now, drag the lymphocyte population from sample A02 onto the existing graph of Sample A01 in the Layout
Editor. When you do that and drop the population, an overlay will be created. You will notice that the graph
border also turns blue, this is to let you know that you are creating an overlay. When you do that and drop the
population, an overlay will be created.

Lesson 6 Fig.21 - overlayed sample

A legend of the overlay will also be present. Left
clicking on the color boxes will provide a color
palette to change colors of selected populations.

With histogram overlays, you can also right

click on the color box to change the line weight,
style or fill.

Lets overlay all lymphocyte populations from

all samples onto this overlay now.
Lesson 6 Fig.22/23 - menus

Go back to the workspace and select the lymphocyte

population in sample A01.

Lesson 6 Fig.24 - selected node

Now, click ‘Edit’ tab and ‘Select Equivalent Nodes’:

This will select all the lymphocyte populations in every sample
in the workspace. You can now drag them all over with one
motion onto the existing plot to overlay every lymphocyte Lesson 6 Fig.25 - all samples overlayed
population.

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Now, hold down the alt key on the keyboard, right click on the color boxes of the legend and select ‘filled’.

Now all the layers of the overlay

should be filled.

You can change the order of the

populations in the overlay by
clicking any row in the legend
and dragging it up or down.

Lesson 6 Fig.26 - coloring option

Histogram overlays can also be offset. Right click on the histogram

overlay and choose offset. Select the ‘Stagger Offset’ option here:

Lesson 6 Fig.27 - all samples filled

By grabbing the back corner of the

staggered offset overlay, you can change
the perspective.

To edit the legend, double click on it.

In the Legend tab of the Graph Definitions

dialog, you can add additional statistics or
keywords. Add the TUBE NAME keyword
by clicking on the key icon.

Lesson 6 Fig.29 - stagger offset

Lesson 6 Fig.28 - menu options

Lesson 6 Fig.30 - graph definitions

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Saving

There are several ways to save your analysis. Click on the

FlowJo icon in the top left corner of your workspace to access
these options.

1) Always save your analysis as a Workspace (.WSP) or Archive

File (.ACS). The workspace file will require your data in
order to function and only saves the link to the files. Hence,
if your files move independently of the workspace, you have
to relink every file back to the workspace. We suggest always
saving your workspace in the same folder as your FCS files.

2) A better way to save the workspace is as an Archive (.ACS) file.

This file type will save the analysis AND the data file information
in one big container file (like a .ZIP file). Lesson 6 Fig.31 - saving options

*If hard drive space is a concern, these files will take up a lot more memory due to the fact that the file is duplicating the .fcs
file information. Most of the time this is not a concern, but if these files are 100MB each and you only have 10GB of space
on your hard drive, you can see how this could be a problem! Luckily, most people have 500GB or more and don’t work with
such large data sets.

3) The other option is to save as a Template (.WSPT). If you will run this experiment again, save the analysis as a
template so all of the analysis is ready for the next experiment. A template will preserve your analysis structure, but
remove the data files. When you run your new data, open the template, drop in your new files and presto, all of your
new files are analyzed! You may have to adjust the gates a little, but everything else will be ready for you.

Thank you for taking the time to learn the fundamentals of FlowJo. Our goal is to produce software that is easy to
use, easy to learn and scientifically accurate. This tutorial has only scratched the surface of FlowJo’s powerful features
and functions. Here are a few more things FlowJo can do, which were not covered in this tutorial:

• Compensation - Corrects for overlap from the emission spectra of fluorochromes. As your experiments
become more complex, the importance of accurate and automated compensation becomes more pronounced.

• Transformation - Uses custom biexponential functions to include zero and negative value events on scale,
maintaining a logarithmic distribution of positive cells.

• Derived Parameters - Supports the creation of new parameters through the algebraic combination of
others, including a wide variety of functions and formula structures, analogous to computed columns in a
spreadsheet.

• Calibrated Parameters - Calculate the quantitation of absolute molecules per cell (MESF) using reference
markers with known calibration values.

• Kinetic Analysis - Measure the change in signal of dyes, or ratios of dyes, over time. Generate statistics and
subpopulations based on the subset of cells that have responded to stimulation.

• Cell Cycle Analysis - Computes the percentage of cells in each phase of the cell cycle, based on a dye
measuring the amount of DNA present in the cells (eg. 7AAD, PI, DAPI) FlowJo is the first package to
integrate multidimensional cell cycle analysis, using BrdU or similar markers to differentiate S phase cells.

• Proliferation Studies - Model the frequency of successive generations, and gate cells by the number of
divisions undergone, as measured by intensity of CFSE, or other intracellular dye.

Help menus from any FlowJo window launch a web browser to access a web page describing that topic, giving you
context sensitive help.

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 You can learn more about FlowJo at the links below.

View the entire Reference Manual:

www.flowjo.com/home/manual.html

For a more in depth tutorial, download the 8 Color PBMC Analysis at:
http://www.flowjo.com/home/tutorials/eightcolor.html

Application tech notes:

http://www.flowjo.com/home/tutorials/

We welcome your feedback! [email protected]

Revision date: 16 April 2013

version 10.0.6

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