Flow cytometry can be used for a variety of applications including medical research, diagnostics, and basic science. It allows for precise quantification of multiple antigens on individual cells through fluorescent labeling and detection. Key uses of flow cytometry include cell counting, sorting, analysis of characteristics and function, detection of microorganisms, biomarker analysis, and protein engineering detection. It is a routine technique in research, clinical practice, and clinical trials.

1. This technology has applications in a number of fields,
including molecular biology, pathology, immunology,
virology, plant biology and marine biology. It has broad
application in medicine especially in transplantation,
hematology, tumor immunology and chemotherapy, prenatal
diagnosis, genetics and sperm sorting for sex pre-selection.
Flow cytometry is widely applied to detect sperm cells
abnormality associated with DNA fragmentation in male
fertility assays. Also, it is extensively used in research for the
detection of DNA damage, caspase cleavage and apoptosis.

2. Photoacoustic flow
cytometry is used in the
study of multi-drug-
resistant bacteria (most
commonly MRSA) to
detect, differentiate,
and quantify bacteria in
the blood marked with
dyed bacteriophages.
In neuroscience, co-
expression of cell
surface and intracellular
antigens can also be
analyzed.
In protein engineering,
flow cytometry is used
in conjunction
with yeast
display and bacterial
display to identify cell
surface-displayed
protein variants with
desired properties
In microbiology, it can
be used to screen and
sort transposon mutant
libraries constructed
with a GFP-encoding
transposon (TnMHA), or
to assess viability.

3. The Main Advantage
• The main advantages of flow cytometry over histology and IHC is the
possibility to precisely measure the quantities of antigens and the
possibility to stain each cell with multiple antibodies-fluorophores, in
current laboratories around 10 antibodies can be bound to each cell. This
is much less than mass cytometer where up to 40 can be currently
measured, but at a higher and slower pace.

4. Aquatic research
• In aquatic systems, flow cytometry is used for the analysis of
autofluorescing cells or cells that are fluorescently-labeled with added
stains.
• This research started in 1981 when Clarice Yentsch used flow cytometry to
measure the fluorescence in a red tide producing dinoflagellates
• Marine scientists use the sorting ability of flow cytometers to make
discrete measurements of cellular activity and diversity, to conduct
investigations into the mutualistic relationships between microorganisms
that live in close proximity,and to measure biogeochemical rates of
multiple processes in the ocean

5. Cell Proliferation assay
• Cell proliferation is the major function in the immune system. Often it is required
to analyse the proliferative nature of the cells in order to make some conclusions.
One such assay to determine the cell proliferation is the tracking dye
carboxyfluorescein diacetate succinimidyl ester (CFSE). It helps to monitor
proliferative cells. This assay gives quantitative as well as qualitative data during
time-series experiments

6. • Cell counting
• Cell sorting
• Determining cell characteristics and function
• Detecting microorganisms
• Biomarker detection
• Protein engineering detection
• Diagnosis of health disorders such as blood cancers
Flow cytometry is routinely used in basic research, clinical
practice, and clinical trials. Uses for flow cytometry include:

7. Flow cytometry can be used for cell cycle
analysis to estimate the percentages of a cell
population in the different phases of the cell
cycle, or it can be used with other reagents to
analyze just the S phase.

8. Why flow cytometry is ideal for cell cycle
analysis
1. When stained with a cell cycle reagent, DNA in the cells bind the dye stoichiometrically (in
proportion to the amount of DNA present in each cell). The flow cytometric analysis of cell
count versus linear fluorescence is used to create a histogram of the DNA content
distribution across the steps of the cell cycle (Figure A).
2. There are standard modeling algorithms that can then be employed to determine the
breakdown of cells in the G0/G1 phase versus S phase, G2, or polyploidy state of the cell
population (Figure B).

9. Live-cell cycle analysis stains—Vybrant
DyeCycle stains
• Classic DNA cell cycle stains such as Hoechst 33342 and DRAQ5 for cell cycle
analysis, but most of these have limitations that have to be considered when using
them in an experiment which is why the Invitrogen Vybrant DyeCycle stains for
live-cell cycle analysis were developed.

10. Fixed-cell cycle analysis stains
FxCycle reagents
• We offer classic DNA cell cycle stains such as DAPI, PI, and 7-
AAD for fixed cell cycle analysis, but these reagents do not
cover the full spectrum of laser excitation available.
• The FxCycle reagents offer options for the 405 nm (violet) and
633 nm (red) laser thereby increasing the ability to multiplex
by freeing up the 488 nm and 633 nm lasers for other cellular
analyses such as immunophenotyping, apoptosis analysis, and
dead cell discrimination.

11. • Precise—Accurate cell cycle analysis in living
cells
• Safe—Low cytotoxicity for combining with
additional live cell experiments
• Cell sort compatible—Easily sort cells based
on phase of the cell cycle
• Flexible—Stains available for all common
laser lines (UV, 405, 488, 532, and 633 nm)
• Flexibility—Options for 405 and 633
nm laser excitation to increase
multiplexabilty in cell cycle studies
• Tight CVs—More accurate analysis due
to a narrow emission spectra requiring
very minimal compensation
Vybrant Dye Cycle stains are: FxCycle stains provide:

12. Difference between Live cell analysis
and Fixed cell anaysis
(The easiest, but not only reliable, is by light scatter.)
• Living cells …are turgid, they have a relatively smooth surface and
osmosis makes the pressure inside bigger than outside. If you shoot
a LASER at them, it will bounce off at low angles (be reflected, more
accurately).
• Dead cells …have no internal pressure, osmosis will not work, their
membranes are ruffled and have many holes. LASERS are reflected
at larger angels, scattered more. Just this filter will distinguish dead
from living cells.
You can combine it easily with size distribution, dead cells will
appear smaller on average.