There are many reasons it may be desirable or necessary to analyze cells in the lab, from assessing the effects of a drug or experimental treatment to studying and diagnosing pathological conditions or characterizing cell phenotypes. There are a host of parameters that may be assessed and consequently a number of tools commonly found in the cell biology lab. One such important tool is the flow cytometer.
Download this infographic to explore:
• How flow cytometry works
• What flow cytometry analyses can tell us
• The applications of flow cytometry
From assessing the effects of a drug or
experimental treatment to studying and
diagnosing pathological conditions or
characterizing cell phenotypes, there are many
reasons it may be desirable or necessary to analyze
cells in the lab. There are a host of parameters that
may be assessed and consequently a number of
tools commonly found in the cell biology lab. One
such important tool is the flow cytometer.
This infographic will explore how flow cytometry
works, what it can tell us and its applications.
How does flow
A flow cytometer combines three main
systems, fluidics, optics and electronics.
When individual cells in the fluidic system
pass a laser beam from the optical system,
they scatter light, which is then measured
by detectors and converted to usable data
in the electronic system.
What are the
Flow cytometry helps out in many
areas of science. Let’s consider some
of the common ones.
Ideally, when using multiple
fluorophores, they should be chosen
to have minimal spectral overlap,
however, some correction is often
required. Compensation, utilizing
control samples, is used to correct
Histograms, dot plots and density
plots are then generated from
the collected data, showing cell
populations for each marker.
show a correlation between two
optical values, such as two different
fluorescent labels, normally with
each cell represented by a dot. The
dots representing cells with similar
values cluster together and often
represent certain cell populations.
Assessing cell health and viability
No matter what your field of interest or the research question you want to answer, if you are
working with live cells, it is important to ensure they are happy and healthy to start with (and
are the cells you want) to obtain meaningful and repeatable results. Flow cytometry is therefore
a key basic tool in cell culture labs for ongoing checks of cells in use. This can be particularly
important, for example, when:
• Generating or receiving a new cell line
• Cells have been in long-term storage
• Cells have been passaged many times
• You are isolating cells from tissues or bodily fluids
This helps to set a baseline for any subsequent treatments the cells may be subjected to as well.
Increasingly, studies are addressing features of cells at the single
cell rather than bulk level, to understand variations within cell
populations and spatial distributions, for which flow cytometry is
able to contribute. There has also been work on systems that do
not damage or require cells to be labeled to this end.
Biopharma and drug discovery
Assessing changes in cell health and viability compared
to their baseline status can provide valuable information
on the effects of drug and biopharmaceutical treatments.
Cellular responses such as apoptosis, proliferation and
cytotoxicity can be assessed. Flow cytometry has become
a vital tool in characterization assays and antibody and
phenotypic screening in the discovery and development
pipeline, more recently assisted by advances in highthroughput flow cytometry.
In the clinic
Flow cytometry can assist in diagnosing disease, making
prognoses and therapeutic monitoring.
In oncology, the technique is used to immunophenotype
cells, identifying those with cell surface markers of
leukemias or lymphomas, for example, and enabling them
to be subclassified.
The technique can also be used to identify and separate
hematopoietic stem cells that can be used to repopulate
bone marrow following chemotherapy for blood cancers.
Immunophenotyping can also be used to diagnose
primary and secondary immunodeficiency disorders such
as X-linked (Bruton’s) agammaglobulinemia and human
immunodeficiency virus (HIV) infection respectively.
Prior to organ transplant, it is important to ensure a match
between organ and donor to minimize the risk of rejection,
for which flow cytometry can be employed.
Other areas of utility include monitoring cardiovascular
disease and sepsis.
Flow cytometry, while not traditionally part of the microbiology
toolkit in part due to cost and sensitivity issues with smaller cells,
can be used to identify and quantify microbes without the need
for culture. This has been facilitated by advances in acoustic
focusing, image enhancement and the development of microbespecific stains in recent years and removes the barriers of some
is an important step in data analysis. Here, users can draw lines,
called gates, to select certain cell populations for further analysis.
As a matter of routine, gates are normally included to select for
data from single, viable cells. If multiple cells pass the laser at one
time, false positives may be generated. Dead cells are also liable to
auto fluoresce, which can interfere with results. Additional gates
can then be added to focus on specific cell populations of interest.
very similar to dot plots with
each cell represented by a
dot, can then then be used to
identify the densest areas of cell
populations and translate this
into a color spectrum where red
typically shows the highest cell
densities and blue the least.
are a type of univariate plot showing
the distribution of the number of
cells positive for a single optical
parameter, such as a specific
What can flow cytometry tell us?
There are three main components to flow cytometry output signal, each of which provides information on different aspects of the cell being analyzed.
To waste container
Fluidic system Optical system Electronic system
Cells from sample
The fluidic system contains the cells to
be analyzed in a buffer or water and is
typically pressurized. Before analysis,
the cells must be disaggregated and
suspended so that they pass through the
system one by one. The cells are stained
with dyes or fluorescent probes targeted
to specific cellular components.
The optical system consists of an excitation
laser, which is directed at the cells as they
pass. The light is scattered by the cells and
the resultant light signal is directed by
mirrors, filtered for specific wavelengths and
recorded by detectors, often photodiodes
and photomultiplier tubes (PMTs). Factors
such as cell size and complexity influence
how the light is scattered.
Increases in the number and quality of
the lasers and detectors over the years
have led to an increase in the number of
different markers that can be detected in a
The electronic system converts the
detector signal to a voltage pulse and
finally a digital output. This typically
includes an amplification step.
1 2 3
Forward scatter (FSC)
Light continuing forward past the cell →
Reflects cell size
Light will be bent as it passes the cell, so the
greater the FSC signal, the smaller the cell.
Side scatter (SSC)
Measured at 90° to the path of the light beam →
Reflects granularity/complexity of cell
Cell structures cause light to be refracted as
it passes through the cell, so the greater the
SSC signal, the more complex the cell interior.
Light emitted by fluorophores in/on the
cell when excited by the laser → Indicates
presence of the molecule/structure the
fluorophore is targeted to bind.
Combined, these metrics can provide
Cell cycle analysis
Cell phenotype Emuneration
Proliferation Cell sorting
…and help perform tasks such as
Inclusion of automated sample loaders is helpful for highthroughput analyses.
Fluorescence-activated cell sorters (FACS) can be included
to guide different cell types that are positive or negative
for a particular parameter into different pots after they are
analyzed, rather than a waste container, to allow specific cell
populations to be collected.
Spectral flow cytometers measure the entire spectrum
of a fluorophore rather than just the peak emission, enabling
fluorophores with overlapping spectra to be analyzed
Imaging cytometry incorporates fluorescence microscopy,
enabling cell morphology and other physical changes to be
Relative cell number
Placebo Drug A
One, two, three...
Additional instrumentation can be incorporated to tailor the flow cytometer to a specific purpose.
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