What is flow cytometry (FACS analysis)?

Abstract
Originally developed in the late 1960s, flow cytometry is a popular analytical cell-biology
technique that utilizes light to count and profile cells in a heterogenous fluid mixture. Flow
cytometry is a particularly powerful method because it allows a researcher to rapidly,
accurately, and simply collect data related to many parameters from a heterogeneous fluid
mixture containing live cells.
Originally developed in the late 1960s, flow cytometry is a popular analytical cell-biology
technique that utilizes light to count and profile cells in a heterogenous fluid mixture. Flow
cytometry is a particularly powerful method because it allows a researcher to rapidly,
accurately, and simply collect data related to many parameters from a heterogeneous fluid
mixture containing live cells.
Flow cytometry is used extensively throughout the life and biomedical sciences, and can be
applied in any scenario where a researcher needs to rapidly profile a large population of loose
cells in a liquid media. For example, in immunology flow cytometry is used to identify,
separate, and characterize various immune cell subtypes by virtue of their size and
morphology.
When additional information is required, antibodies tagged with fluorescent dyes, and raised
against highly specific cell surface antigens (e.g. clusters of differentiation or CD markers)
can be used to better identify and segregate specific sub-populations within a larger group.

In a flow cytometer:
Sample cells are passed through a narrow channel one at a time.
Light is used to illuminate the cells in the channel.
A series of sensors detect the types of light that are refracted or emitted from the cells.
Data acquired by the sensors is compiled and integrated to build a comprehensive
picture of the sample.
A flow cytometer uses refracted or emitted light to count and identify cells.

Learn about different types of light used in a flow cytometry experiment in the
following figures.

Forward Scatter
Forward scattered light is refracted by a cell in the flow channel and continues along in the
light path (i.e. The same direction that the light was originally traveling). Forward scattered
light is detected by a sensor in the light path, and is typically used to identify particle size.
Forward scattered light is most commonly used to detect the size of the object in the light
path. Larger objects will produce more forward scattered light than smaller objects, and larger
cells will have a stronger forward scatter signal
Side Scatter

Side scattered light light passes from the illumination source into the flow channel, is
refracted by cells in a direction that is outside of the original light path. Side-scattered light is
detected by a sensor that is orthogonal to the original light path.
Side-scattered light is usually used to make a determination regarding the granularity and
complexity of the cell in the light path. Highly granular cells with a large amount of internal
complexity, like neutrophils, will produce more side-scattered light, and a higher side-scatter
signal than cells with a low-granularity and complexity.

Multiparametric Analysis
A mock flow cytometry dot-plot, plotting forward vs side-scattered from a population of
leukocytes. Cell populations are marked by their probable identity:
D Presumed debris, very small items with low low forward- and side- scatter.
L/M Probable leukocytes/monocytes, small to medium cells with low internal
complexity/granularity. These cells generate a medium forward-scatter and low side-scatter
signal intensity
G Probable granulocytes, large cells with high internal complexity/granularity. These cells
generate high forward- and side-scatter signals.
While some identities can be confirmed by forward and side-scatter profiles, labeling with a
cell-type specific marker always provides greater resolution and certainty when profiling
complex heterogeneous populations of cells.
For example, in the plot above, a researcher may be able to distinguish between granulocytes
and lymphocytes using forward and side-scattered light. However, three classes of
granulocytes (neutrophils, basophils, and eosinophils) are very similar in size and structure,
giving them similar light-scattering properties. In this instance, neutrophils could be
selectively labeled by virtue of their expression a neutrophil specific marker like ELANE.

Fluorescence Emission

Fluorescent light is emitted by fluorescent molecules after excitation by a compatible

wavelength laser. Fluorescent light may originate from naturally fluorescing materials in the
cell, or may originate from fluorescent dyes or fluorescence-tagged antibodies that have been
used to label a specific structure on the cell.
FACS: sorting cells based on flow cytometry data

The terms flow cytometry and fluorescence-activated cell sorting (FACS) are often used
interchangeably. In practice, there are differences between the two methods.
FACS is a derivative of flow cytometry that adds an exceptional degree of functionality.
Using FACS a researcher can physically sort a heterogeneous mixture of cells into different
populations.
By using highly specific antibodies tagged with fluorescent dyes, a researcher can perform
FACS analysis and simultaneously gather data on, and sort a sample by a nearly limitless
number of different parameters.

In a FACS experiment:
Forward-scatter, side-scatter, and fluorescent data is collected, as in conventional flow
cytometry.
User-defined parameters provide information on how cells should be sorted.
Based on these parameters, the FACS machine uses an electrode to impose an
electrical charge on each cell.
Upon exiting the flow chamber, electromagnets will sort cells by charge into separate
vessels.

What does flow cytometry data look like?

In a flow cytometry experiment, every cell that passes through the flow cytometer and is
detected will be classified as a distinct event.
Additionally, each type of light that is detected by the flow cytometer (forward-scatter, side-
scatter, and each wavelength of fluorescence emission) will be assigned its own unique
channel. Flow cytometry data will plot each event independently, and will represent
the signal intensity of light detected in each channel for every event.
Flow cytometry data is typically represented in one of two ways: histograms, which measure
or compare only a single parameter, and dot-plots which compare 2 or 3 parameters
simultaneously on a two- or three-dimensional scatter-plot.
A histogram typically plots the intensity detected in a single channel along one axis and the
number of events detected at that intensity is in a separate axis. A large number of events
detected at one particular intensity will be displayed as a spike on the histogram.
By contrast, in a dot plot, each event is represented as a single point on a scatter-plot.
Intensity of 2 different channels (or 3 different channels in a three-dimensnal plot) are
represented along the various axes. Events with similar intensities will cluster together in the
same region on the scatter-plot.
Histogram data from product ABIN118256

Cells were stained using anti-CD90 antibody ABIN118256. This histogram plots a single
parameter (FITC intensity, horizontal axis) against the number of events detected (vertical
axis).
Negative control cells are plotted in blue.
Experimental cells that express the antigen (CD90) are plotted in red.
ABIN192135

Cells were stained using anti-CD48 antibody ABIN192135. This dot-plot plots two
parameters simultaneously. Side-scatter intensity (vertical axis) is plotted against against
fluorescence intensity in the APC channel (horizontal axis). Each distinct event is represented
as a single dot.>
Note: In dot-plot data, large samples will often result in a heavy cluster of events represented
in the same region of the plot. There are many methods for adding additional resolution to
these regions. For example, a heat map, as in the example above, may be used to provide
information about event density in a given region of the plot.
Histograms and dot-plots both provide different advantages for flow cytometry data analysis.
Choosing how best to represent your data can help ensure that it tells a complete story in a
simple, comprehensible format.
Historgrams
Are fast to read and easy to understand.
Are most useful when only one parameter (e.g. intensity from a single fluorescent
channel) is important.
Usual representation includes the intensity of a single channel (horizontal axis) vs
number of detected events (vertical axis).
Multiple overlaid histograms can be used to compare a single parameter from two
different sample populations (e.g. experimental vs. control).
Dot-plots
Are most useful when you need to compare multiparametric data (e.g. Intensity of
side-scatter vs forward-scatter channels).
Can be two- or three-dimensional
Intensity of each channel is represented on its own axis.
Each distinct event is represented at a single dot.
Are a more complex, more illustrative representation of data.
Dot-plots and histograms are not mutually exclusive, and most complex flow cytometry
experiments will make use of multiple plots to display rich, multi-parametric data on a
sample.
In many instances, more than three parameters need to be plotted simultaneously. In this case,
a data-analysis technique known as gating can help to give additional resolution and
flexibility, allowing for analysis of a nearly limitless quantity of parameters simultaneously
across several different scatter-plots and histograms.
Gating adds resolution to flow cytometry data
In short, gating is a method for selecting cells from a flow cytometry experiment that you
want to analyze in more specific detail. Gating allows a researcher to gather and display more
information about a subpopulation of cells than could normally be displayed on a 2- or 3-
dimensional dot-plot.
Gating adds resolution to a flow cytometry experiment, and allows for simultaneous analysis
of a nearly limitless number of different parameters (channels).
In a gated flow cytometry experiment:
A user collects flow cytometry data from one or more channels on a dot-plot.
Based on the data acquired, the user draws a gate box selecting a subpopulation of
cells for further analysis.
 The subpopulation of cells within the gate will be specifically highlighted on other
plots displaying information from alternate channels.
Gates add an incredible amount of flexibility to flow cytometry, granting up to single-cell
resolution for each channel available to the researcher. Multiple gates can be established for a
single scatter-plot, and gates can be "stacked" and combined (i.e. A subpopulation of cells
gated for in channels 1 and 2 can be further gated for channels 3 and 4 to allow for more
specificity and deeper analysis).
An example of gating

A mock example of gating. In the image two subpopulations of cells are gated based on their
forward and side-scatter intensity profiles.

The same population of cells are highlighted by a cohesive color scheme when analyzing two
different channels measuring fluorescent intensity in the image above.
A note on compensation
Spillover from one channel into another one can cause falsely as positive identified signals.
Spillover is the artefactual signal that one fluorescent dye can cause in the channel of another
fluorophore as a function of its relative brightness and emission spectrum. This is where
compensation comes into play. Compensation is a procedure that isolates the signal from one
particular channel from the other channels used in the same experiment. FITC e.g. has its
emission peak in the green range of the electromagnetic spectrum. It does however also
fluoresce in the yellow channel where PE emits light. Simply put, compensation subtracts the
FITC signal from the PE channel.
This signal in the wrong channel is subtracted from the signal caused by the fluorophore of
interest. In the above sample, the ratio of the green and the yellow component at a given
excitation wavelength is constant for FITC. It is thus possible to infer the amount of
unspecific yellow FITC signal in the PE channel from the measurement in the green channel
using the constant compensation coefficients. The same is true for the spillover from the PE
into the FITC channel.
This signal in the wrong channel is subtracted from the signal caused by the fluorophore of
interest. In the above sample, the ratio of the green and the yellow component at a given
excitation wavelength is constant for FITC. It is thus possible to infer the amount of
unspecific yellow FITC signal in the PE channel from the measurement in the green channel
using the constant compensation coefficients. The same is true for the spillover from the PE
into the FITC channel.