2. Index
• History
• What is flow cytometry
• Principles
• Data analysis
• Application
• Flow cytometry bioinformatics
• Advantages
• Disadvantages
• Flow cytometry and next generation sequencing
• New advance in flow cytometry
3. History
• Flow cytometry was invented in 1953 by Wallace H. Coulter.
• The original name of the fluorescence-based flow cytometry technology was
"pulse cytophotometry" .
based on the first patent application on fluorescence-based flow cytometry.
4. What is flow cytometry
• It is a technique used to count and analyze the size, shape and properties of an
individual cell within a population of cells.
• Tens of thousands of cells can be quickly examined and the data gathered are
processed by a computer.
• Flow cytometry is routinely used in basic research, clinical practice, and clinical
trials.
5. What is flow cytometry
• A flow cytometer is similar to a microscope, except that, instead of producing
an image of the cell,
flow cytometry offers high-throughput, automated quantification of specified
optical parameters on a cell-by-cell basis.
6. What is flow cytometry
• Component:
A flow cytometer has five main components:
1. Flow cell (Fluidics)
2. Optic system
3. Detector
4. Amplification system
5. Computer for analysis of the signals
7. What is flow cytometry
• Component:
1. Flow cell
has a liquid stream, which carries
and aligns the cells so that
they pass single file through
the light beam for sensing.
8. What is flow cytometry
• Component:
2. Optic system (lasers)
consists of lasers to illuminate the particles in the sample stream and optical
filters to direct the resulting light signals to the appropriate detectors.
9. What is flow cytometry
• Component:
3. Detector
converts analog measurements of forward-scattered light (FSC) and side-
scattered light (SSC) as well as dye-specific fluorescence signals into digital
signals that can be processed by a computer.
11. What is flow cytometry
• The current record for a commercial instrument is ten lasers and 30
fluorescence detectors.
• Increasing the number of lasers and detectors allows for
1. multiple antibody labeling
2. can more precisely identify a target population by their phenotypic markers
Fluorescein isothiocyanate
phycoerythrin
12. What is flow cytometry
• Component:
4. Amplification system
Amplify the signals (photons) and converted to a voltage pulse.
13. What is flow cytometry
• Component:
5. Computer to analyze the signals
14. What is flow cytometry
• Flow cytometry dyes:
1. Classic DNA dyes
They are the first type of live dead cell dyes that most scientists and flow
cytometrists consider for their experiments.
Examples of these dyes include:
the Sytox dyes
DRAQ7
propidium iodide (PI)
and 7-aminoactinomycin D (7-AAD).
15. What is flow cytometry
• Flow cytometry dyes:
1. Classic DNA dyes
Classic DNA dyes are easy to use and typically added at the end of staining,
which means they require minimal incubation.
These DNA binding dyes are also inexpensive.
16. What is flow cytometry
• Flow cytometry dyes:
2. Amine dyes:
Two of the most commonly used amine reactive dyes are:
ViViD and Aqua Blue
Amine dyes are membrane impermeant, but rather than binding DNA, they work
by binding the amine groups of cellular proteins.
17. What is flow cytometry
• Flow cytometry dyes:
3. Vital dye:
Instead of binding to DNA, like the classic DNA dyes, or to protein like the amine
reactive dyes, this third class of reagents measures viability by fluorescing when
acted upon in metabolically active cells.
Example:
Calcein acetomethoxy
18. What is flow cytometry
• Flow cytometry dyes:
4. Single dyes:
Such as: FITC, PE, APC and PerCP .
19. What is flow cytometry
• Flow cytometry dyes:
5. Tandem dyes:
Tandem dyes comprise a small fluorophore covalently coupled to another
fluorophore.
Tandem dyes are very useful for multicolor fluorescence studies, especially in
combination with single dyes.
For example:
Alexa Fluor 488, phycoerythrin (PE), peridinin chlorophyll protein (PerCP)-Cy5.5
and PE-Texas Red
20. What is flow cytometry
• Flow cytometry dyes:
6. Fluorescent Proteins:
for understanding protein expression.
The benefit of these fluorescent proteins is the quantitation of intracellular
markers in live cells without requiring permeabilization of the cell membrane
Examples:
green fluorescent protein (GFP)
mCherry
yellow
fluorescent protein
CFP
21. Principles
1. Sample Preparation:
• The primary requirement for all types of flow cytometric analysis is that the
cells under analysis must be in a single-cell suspension. It is very important to
obtain a single cell suspension to avoid clogging up the system with clumps.
• Any suspended particle or cell from 0.2–150 micrometers in size is suitable for
analysis. Cells from solid tissue must be desegregated before analysis.
22. Principles
1. Sample Preparation:
• Peripheral blood mononuclear cells (PBMCs) isolated from whole blood
through Ficoll gradient centrifugation, or RBC lysed whole blood, or non-
adherent cultured cells are readily applicable for flow cytometric analysis.
• Adherent cultured cells or cells present in solid organs should first be made
into a single cell suspension before flow analysis by using enzymatic digestion
or mechanical dissociation of the tissue, respectively.
23. Principles
2. Antibody Staining:
• Each human cell expresses hundreds of thousands of cell surface antigens that
specify their cell type, biological function, development stage, and more.
• We can utilize antibodies specific for these surface markers to analyze these
cells directly by flow cytometry.
25. Principles
4. The machine will absorb the suspension sample and mix the sample into
stream of saline solution and lead the cells into narrowing channel, causing
the cells to form a single file line.
26. Principles
5. Each cell within the sample moves across the laser beam, allowing each cell
within the sample to be individually analyzed.
As each cell passes through the laser beam the laser beam will scattered in
multiple directions
27. Principles
• Scattered light can be either:
• Forward scatter (for cell size)
• or side scatter (for cell granularity)
28. Principles
6. The flow cytometer will detect the light in a forward manner, called forward
scatter. The light on the side is called side scatter.
• Forward scatter:
• The amount of forward scatter light for each cell is detected by a detector on
the far side of the cell from the laser
Forward scatter
side scatter
30. Principles
• The detector convert the scattered light into voltage pulse.
which directly proportional to the amount of forward scattered light.
Forward scatter detector
31. Principles
• The computer converts these data into histogram blot with the amount of
forward scattered light on the X-axis and the number of cells on the Y-axis.
32. Principles
• side scattered:
• The amount of side scattered light is detecting is detected by a detector
located vertically to the path of the laser beam.
side
scatter detector
34. Principles
• The flow cytometer convert the detected side scattered light into voltage
pulse, which directly proportional to the amount of side scattered light.
36. Principles
• In addition to analyze the cell’s shape, size and complexity, flow cytometry can
also detect emitted lights from from excited fluorescence molecules.
• Such as: antibodies, dyes and stains.
37. Principles
• Labeled samples flow cytometry:
1. The florescent molecules or (fluorophores) are excited by laser’s wave
length, when the cells passes through the laser beams.
2. After excitation, the light emitted by the fluorophores is directed in a path
with emittion filters that allow for detection of multiple different
fluorophores emitting light in a cell.
3. Flow cytometry can detect the presence the fluorophores and also quantify
the relative amount of a fluorophores within a cell.
39. Principles
• Flow cytometer can also be used to detect light emitted by multiple
fluorophores in the same sample.
• While the flow cytometer is allowed to detect 2-4 colors at the same time,
some flow cytometers can detect up to 18 colors at the same time
40. Principles
• Data can be shown in a different types of data displays:
1. Frequency distribution (Histogram)
2. Dot plot
3. Density plot
4. Contour plot
41. Data analysis
1. Frequency distribution (Histogram):
• These plots show the intensity of expression versus the number of events
(particles or cells).
• Histograms are useful for cell cycle and proliferation analysis.
42. Data analysis
2. Dot plot:
Used between two parameters, usually size and granularity.
Small sized
Less
granules
44. Data analysis
3. Density plot:
a way to show not just expression levels, but the relative number (i.e. density) of
events in a given region.
45. Data analysis
4. Contour plot:
Contour plots display the relative frequency of the populations, regardless of the
number of events collected.
46. Applications
• The technology has applications in a number of fields including:
• Molecular biology
• Pathology
• Immunology
• Virology
• Plant biology
• Marine biology
47. Applications
• It has broad application in medicine especially in :
• Transplantation
• Hematology
• Tumor immunology and chemotherapy
• Prenatal diagnosis
• Genetics
• Cancers
48. Applications
• Flow cytometry in genetics:
1. Detect sperm cells abnormality associated with DNA fragmentation in male
fertility assays
2. Used in research for the detection of DNA damage
3. caspase cleavage
4. Apoptosis
The measurement of cellular DNA content by flow cytometry uses fluorescent
dyes, such as propidium iodide, that intercalate into the DNA helical structure.
5. The fluorescent signal is directly proportional to the amount of DNA in the
nucleus and can identify gross gains or losses in DNA
6. Abnormal DNA content, also known as “DNA content aneuploidy”, can be
determined in a tumor cell population
49. Flow Cytometry Bioinformatics
• Flow cytometry bioinformatics:
• is the application of bioinformatics to flow cytometry data, which involves
storing, retrieving, organizing and analyzing flow cytometry data using
extensive computational resources and tools.
• Flow cytometry and related methods allow the quantification of multiple
independent biomarkers on large numbers of single cells.
• In the 2000s, the creation of a variety of computational analysis methods, data
standards, and public databases for the sharing of results has started.
50. Advantages
1. Accurate (It also takes off any debris or dead cells when providing the final
data).
2. High specificity for discrete cell subsets and rare populations.
3. Rare cell subsets with frequencies as low as 0.01% (e.g., antigen-specific
cytokine-producing cells) can be detected.
4. Flow cytometric cell sorting allows for isolation of cells of interest to very
high purity
51. Disadvantages
1. Flow cytometry is limited by its requirement that analyzed cells be in
suspension, making information on tissue architecture and cell–cell
interactions unavailable.
2. Flow cytometry may generate massive amounts of data, making analyses
complicated.
3. lack of standardization in assay and instrument set-up; standards are also
lacking in how flow data are analyzed and reported.
4. Expensive.
52. Flow cytometry and next generation
sequencing
• Flow cytometry is a method that is increasingly being integrated with other
technologies to provide richer data for understanding cellular makeup and
function.
• In particular, it is used in conjunction with next-generation sequencing
applications such as RNA sequencing (RNA-seq).
• The integration of cell-sorting technologies, such as index sorting, allows a
finely detailed understanding of the cell receptor phenotype for each single
cell in an RNA-seq application.
53. New Advances
• Spectral flow cytometry:
(Recent advances in optics and detectors have made it feasible to make full
spectral measurements on the sub-millisecond time scales)
54. New Advances
• Imaging flow cytometry:
(unique capability of identifying collected events by their real images)
55. New Advances
• Computational flow cytometry:
allow scientists to measure an increasing number of parameters per cell,
generating huge and high-dimensional datasets.
allows the automation of population identification, biomarker discovery and
predictive modelling to highlight potentially new and interesting cell types that
correlate with clinical outcomes.
56. Conclusion
• Flow cytometry is a sophisticated instrument measuring multiple physical
characteristics of a single cell such as size and granularity.
• Its working depends on the light scattering features of the cells under
investigation, which may be derived from dyes or monoclonal antibodies
targeting either extracellular molecules located on the surface or intracellular
molecules inside the cell.
• flow cytometry a powerful tool for detailed analysis of complex populations.
