FLOW CYTOMETRY/

FLUORESCENCE
ACTIVATED CELL SORTING
Dr. Neha Arya
Translational Medicine
AIIMS Bhopal

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 Introduction
■ Flow cytometry is defined as the simultaneous measurement of multiple physical
characteristics of a single cell as the cell flows in suspension through a measuring device.
■ The fluorescent antibody techniques (immunofluorescence) described are extremely valuable
qualitative tools, but they do not give quantitative data.
■ This shortcoming was remedied by development of the flow cytometer, which was designed to
automate the analysis and separation of cells stained with fluorescent antibody.
■ ‘Flow Cytometry’ as the name suggests is a technique for cell counting and measurement of
different properties of the cell (‘cyto’= cell; ‘metry’=count/measurement).
■ It is a laser based technology that measures and analyses different physical and chemical
properties of the cells/particles flowing in a stream of fluid through a beam of light.

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 Flow Cytometry Compared With
Other Techniques
■ The original flow cytometer was the Coulter counter (Beckman Coulter M) invented by
Wallace Coulter in the 1950s.
■ All current automated, non–slide-based hematology instruments are flow cytometers.
■ The Coulter principle uses the resistance of a cell in an electrical current as it passes
through an orifice to determine numbers of particles and the size of the particle.
■ Flow cytometry has specifically come to denote the use of fluorescence measurement,
usually with a laser light source. In laser flow cytometers, light scatter is used to measure
the intrinsic size and granularity of the cell. In addition, fluorescence can be used to
measure extrinsic features such as specific protein expression and nucleic acid content
using added reagents, such as fluorescent stains and antibodies.

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 By using appropriate antibody panels, flow
cytometry can reveal
■ Cell type (hematopoietic, lymphoid, or nonhematopoietic),

■ Cell lineage (B- and T cells, natural killer cells, myeloid/ monocytic cells,
neuro/neuroendocrine cells, and epithelial cells),

■ Cell maturation stage (precursors vs. matured cells)

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 Principle
■ Flow cytometry involves the analysis of the optical and fluorescence
characteristics of single particle (e.g. cells, nuclei, chromosomes) during their
passage within a narrow, precisely defined liquid stream.
■ The flow cytometer uses a laser beam and light detector to count single intact
cells in suspension.
■ Every time a cell passes the laser beam, light is deflected from the detector, and
this interruption of the laser signal is recorded.
■ Those cells having a fluorescently tagged antibody bound to their cell surface
antigens are excited by the laser and emit light that is recorded by a second
detector system located at a right angle to the laser beam.
■ The simplest form of the instrument counts each cell as it passes the laser
beam and records the level of fluorescence the cell emits; an attached
computer generates plots of the number of cells as the ordinate and their
fluorescence intensity as the abscissa.

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 Principle
■ FACS is capable of sorting populations of cells into different
containers according to their fluorescence profile.
■ Use of the instrument to determine which and how many members of
a cell population bind fluorescently labeled antibodies is called
analysis; use of the instrument to place cells having different patterns
of reactivity into different containers is called cell sorting.
■ The parameters analyzed by flow cytometry include:
– physical properties of cells; the size, cytoplasmic granularity, and
amount of DNA contents; and
– cell antigens/markers (surface, cytoplasmic, and nuclear) that
can be recognized by specific antibodies.

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 Three Major Components

■ Fluidics: Transports the cells to the laser interrogation point

■ Optics: Collects light signal generated by scatter and

fluorescence emission

■ Electronics: Converts optical signals into digital signals that

can be processed by a computer

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 Fluidics

■ The flow cytometer has 2 sources of fluid passing through it: a

sheath fluid that is usually a buffered saline, and the
specimen containing the cells to be analyzed.
■ The specimen forms a core, which is injected into the middle
of the sheath fluid, and both pass through the flow chamber.
■ The outer sheath fluid causes hydrodynamic focusing, which
compresses the specimen core into a small area. Cells are
forced to pass singly through the focal point of the laser, which
is crucial for collecting accurate information about each cell in
the specimen.

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 Optics
■ A laser beam is used in most flow cytometers as the light source to
excite a fluorescent tag (eg, a fluorescent antibody) bound to the cells
in the specimen.
■ Light Source: Argon Laser, Red-helium Neon Laser
■ Before striking the cells, the light from either source passes through
focusing and shaping optics to form an elliptical beam with a broad,
maximal intensity in the center.
■ The beam then passes through the center of a flow chamber, which is
a quartz cuvette with an approximately 200-μm inner diameter. The
specimen is also injected through the middle of this flow chamber,
where the laser beam strikes the cell

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 SCHEMATIC DIAGRAM OF A FLOW
CYTOMETER

PMT-photomultiplier tubes
ADC-analogue-to-digital converter
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 Basic Flow Cell
Sample: Inner Stream containing a mixed population of cells

Sheath Fluid: Outer Stream

Sensing Area

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 Light Scatter Parameters

■ When the laser strikes the cell, light is diffracted around the edges of the cell,
producing a diffraction pattern along the path of the laser beam.
■ This scattered light is approximately equivalent to the cell circumference and is
the same wavelength as the exciting laser light (usually blue).
■ Forward Scatter (FCS) is an indicator of cell size, shape and refractive index
which is related to cell membrane integrity
– is proportional to the surface area or size of a cell.
■ Side Scatter (SSC) is an indicator of cellular granularity or cellular inclusions
– is proportional to the granularity or internal complexity of a cell
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 Forward Scatter
FSC or FALS (Forward Angle Light Scatter)

FSC signal is relative to

cell size and refractive
index which is related to
cell membrane integrity

Laser Excitation Photodiode FSC Detector

Termed forward scatter (FSc) or

forward angle light scatter (FALS), it is
collected along the same axis as the laser
beam.
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 Side Scatter
SSC or RALS (Right Angle Light Scatter( or 90LS (90-degree light scatter)

SSC is proportional to the internal

Blue laser light is also reflected off complexity of the cell.
the cell and off internal structures in The greater the number of
the cell. inclusions or granules, the higher
the SSC.

Laser Excitation

Termed side scatter as it is

detected by a collection objective set at
SSC PMT Detector – 90 Degrees to FSC Detector
90 degrees to the incident laser light.
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 Dot Plot

A three part differential is possible by viewing FSC vs. SSC in peripheral blood

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 Optics (contd.)
■ Fluorescent probes can also be added to the cell for detecting specific molecules in the cell.
These probes are typically antibodies to cellular antigens with a covalently attached
fluorochrome.
■ The fluorochrome, excited by the laser light, will fluoresce at a longer wavelength than that of
the excitation light.
■ The amount of light that is scattered by the cell or particle gives information relative to it’s
size and internal structures.
■ Similar to side-scattered blue all of these fluorescent signals pass through the objective set at
90 degrees to the incident laser light.
■ Additional dichroic mirrors reflect specific wavelengths of light, allowing separation of the
signals from each fluorochrome with minimal overlap.
■ In front of each photomultiplier, a final bandpass filter precisely defines the wavelength of
light entering that detector.
■ The fluorescence of specific markers conjugated to fluorescent dyes is distinguished by light
detectors called photomultipliers tubes. These tubes are used to amplify the weak light signal
from a fluorescent marker.

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 LASERS

■ Light Amplification by Stimulated Emission of Radiation

■ The laser emits light in the form of electromagnetic radiation at a

single wavelength known as monochromatic light

■ Lasers used for flow cytometry range from 300nm to 700nm

excitation.

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 Colors

■ 488 nm wavelength is the most commonly used type of laser in Flow Cytometers
■ Many 5 laser instruments have these additional lasers
– 355 nm UV
– 405 nm Violet
– 640 nm Red
– 561 nm Yellow-Green

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[Link]
 Immunophenotyping analysis needs

■ Antibodies
■ Fluorochromes
– Fluorochromes are substances that can be excited by certain light source
(such as laser) and emit a fluorescent signal at a single wavelength.
– Fluorescent dyes can directly bind to certain cellular content, such as DNA
and RNA, and allow us to perform quantitative analysis on individual cells.
– However, in most cases fluorochromes are conjugated with monoclonal
antibodies, which specifically target cellular antigens/markers.

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 Characteristics of fluorochromes
commonly used in flow cytometry
FLUOROCHROMES CONJUGATED TO ANTIBODIES EXCITATION WAVELENGTH(NM) EMISSION WAVELENGTH(NM)

Fluorescein isothiocyanate (FITC) 488 530

Phycoerythrin (PE) 488 580

PE-Texas Red 488 615

PE-Cy5 488 670

Peridinin chlorophyl protein(PerCP) 488 670

Allophycocyanin (APC) 633 670

APC-Cy7 633 767

Interestingly, although some of them can be excited by the same light source, the
different fluorochromes may emit fluorescent signals with different wavelengths/colors.
Thus, multiple fluorochromes can be simultaneously excited by a light source and
detected by their emission fluorescent signals with different wavelengths, respectively.
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 Immunophenotyping
■ Antibodies conjugated to fluorescent dyes can bind specific proteins on cell
membranes or inside cells.
■ When labeled cells are passed by a light source, the fluorescent molecules are
excited to a higher energy state.
■ Upon returning to their resting states, the fluorochromes emit light energy at higher
wavelengths.
■ The use of multiple fluorochromes, each with similar excitation wavelengths and
different emission wavelengths (or “colors”), allows several cell properties to be
measured simultaneously.

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 Simultaneous detection of multiple cell
antigens/markers.

Multiple cell antigens ( Ag ) are recognized by fluorochromeconjugated specific antibodies ( Ab ). Because different
fluorochromes have different emission wavelengths/colors, they can be simultaneously detected by a flow
cytometer.
FITC
Not forfluorescein isothiocyanate;
dissemination, please! PE phycoerythrin; PerCP peridinin chlorophyll protein; PE-T Red PE-Texas Red .
 Electronics
■ The Photomultiplier tubes (PMT) detectors collect photons of light and
convert them to electrical signals that can be amplified using log
amplifiers.
■ Linear amplifiers are used to amplify the forward angle and side
scatter signals. These amplified electrical signals are then analyzed
and recorded.
■ The voltages are converted into numbers which can be further
analyzed. This process is known as an analog-to-digital (A-D)
conversion.
■ The information from each cell that passed through the laser beam is
now in a form that can be analyzed using various software programs
designed specifically for flow cytometry data.

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 Compensation
■ Spectral overlap between dyes results in the detection of the
fluorescence from one dye in one or more other detectors
(primary vs. spillover fluorescence).

■ Is used to electronically subtract the fluorescence emission

spectral overlap that can occur between different probes.

■ Data that is under compensated will lead to false positives.

■ Data that is over compensated will lead to an under estimate of

positives.

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 Data Analysis

■ Dot Plots
■ Histograms are a common and reliable
method used to present flow data for analysis.

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 Data Analysis
FITC Positive
PE Negative

FITC FITC Positive

PE Positive

PE Positive
FITC Negative

PE

Negative for PE and FITC

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 Gating
• To optimize the analysis of multiparameter experiments, gating
is performed to isolate subpopulations of interest
• Gating is in essence electronic window that sets upper and
lower limits on the type and amount of material that passes
through.
• It is used to separate a sub-population from heterogeneous
population.
• It permits very specific questions to be asked about a particular
population.

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 Example of Gating
Exclude Debris

Exclude Doublets
Analysis on single cells only!

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 Example of Gating
Living Cells - DAPI

Dead Cells

Live Cells

Epithelial cells CD45-

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 Example

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 Fraction of live and dead cells in the STEMs as function of Fluorescent micrographs (A) and fluorescence-
time as assessed by fluorescence activated cell sorting activated cell-sorting histogram (B) demonstrating
experiment uptake of glycosaminoglycan-functionalized poly-
lactide-coglycolide
(PLGA) nanoparticles by lung epithelial
adenocarcinoma (A549) cells
Lamichhane
Not and Arya,
for dissemination, BMC Cancer 2016
please! Lamichhane and Arya, International Journal of nanomedicine 2015
 Gating strategies

■ Gating Strategy #1: Forward and side scatter density plots for identifying your cell
population of interest and excluding debris
■ Gating Strategy #2: Forward scatter height versus forward scatter area density plot
for doublet exclusion.
■ Gating Strategy #3: Single parameter histograms for identifying cells with a
particular marker expression
■ Gating Strategy #4: Two parameter density plots for further analysis

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 Flow Sorting
■ Flow sorting selects specific cells or particles based on any number of parameters
and physically isolates them.
■ One of the major differences between a sorter and an analyzer is the ability of the
flow cell to vibrate by means of a piezoelectric crystal at a frequency 20,000 Hz or
higher.
■ The vibration causes the stream to form droplets. Each droplet generated is of the
same size.
■ Each cell is analyzed as it passes through the flow cell. If the cell meets the criteria
established for sorting, a voltage is applied to the stream at the moment that a
droplet is forming. The drop will then be charged and deflected by the high voltage
deflection plates as it moves downward.
■ The voltage on the stream is reduced to zero to avoid charging unwanted drops.
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 Flow Sorting

Deflection Plates (6,000 volts)

Graded Test
Graded Test
Graded Test Graded Test
Tube Clip Art
Tube ClipGraded
Tube Clip Art Art Test Graded Test
Tube Clip Art
Tube Clip Art Tube Clip Art

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Not for dissemination, please!
[Link]
 Process of FACS

■ A fluorescence-activated cell sorter provides the ability to separate cells identified

based on size and/or fluorescence.
■ Droplet based cell sorters first analyze the particles, but also have hardware that
can generate droplets and a means of deflecting or directing droplets containing the
desired particles into a collection tube.
■ Droplets can be formed by using high-frequency (cycles/second, Hz) vibration of the
nozzle at an optimal amplitude (in volts).
■ This is typically created by a piezoelectric crystal.
■ As cells pass through the laser interrogation point, light emitted from the cell is
collected via detectors, and this signal is then processed by the instruments
electronics which then tells the instrument to apply a charge to the droplet.
■ The charged droplet can then be deflected into the appropriate tube for collection.

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 Sort Process

– Cell to be sorted enters the stream and is identified based on scatter and or
fluorescence criteria.
– Cell triggers the lasers
– Cell moves down the stream
– Cell enters the last drop before breakoff
– Stream is charged
– Drop containing the cell of interest separates from the stream and carries a charge
– Stream is grounded
– Charged drop enters electric field and is deflected
– Cell is collected in a vessel containing a buffer

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 Applications
Immunophenotyping Cell Sorting Cell Proliferation
Cell subsets are The cell sorter is a Proliferation can be measured
measured by labeling specialized flow cytometer by labeling resting cells with a
population specific with the ability to physically cell membrane fluorescent dye
isolate cells of interest into e.g. (CFSE) and measuring the
proteins with a
reduction of the fluorescence
fluorescent tag on the cell separate collection tubes.
signal.
surface.

Cell Cycle Analysis DNA Content Apoptosis

Flow cytometry can analyze Fluorescent dyes, such as The two distinct types of
replication states using propidium iodide, intercalates cell death, apoptosis and
into the DNA helical structure.
fluorescent dyes to necrosis, can be
The fluorescent signal is
measure the four distinct directly proportional to the assessed by flow
phases of the cell cycle. amount of DNA in the nucleus. cytometry.

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 Propidium iodide (PI) - Cell Viability
How the assay works:
■ PI cannot normally cross the cell membrane
■ If the PI penetrates the cell membrane, it is assumed to be
damaged
■ Cells that are brightly fluorescent with the PI are damaged or dead

Viable Cell Damaged Cell

PI
PI
PI PI PI
PI PI

PI

PI
PI
PI

PI

PI PI

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 DCFH-DA DCFH DCF
2’,7’-dichlorofluorescin diacetate
O O
CH3-C-O O O-C-CH3

Cl Cl 2’,7’-dichlorofluorescin
H
COOH HO O OH
Fluorescent
Cellular Esterases Cl Cl
H 2’,7’-dichlorofluorescein
COOH
Hydrolysis HO O O
H2O2
Cl Cl
H
DCFH-DA Oxidation COOH

Neutrophils

DCFH-DA
Monocytes 80

Control PMA-stimulated PMN

60
DCFH H O

counts
2 2
Lymphocytes 40

DCF 20

0
.
1
log FITC 10
1
Fluorescence
100 100 0

Neutrophil Function, Oxidative Burst

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 Phagocytosis
■ Uptake of Fluorescent labeled particles
■ Determination of intracellular or extracellular state of
particles
How the assay works:
• Particles or cells are labeled with a fluorescent probe
• The cells and particles are mixed so phagocytosis takes place
• The cells are mixed with a fluorescent absorber to remove fluorescence from
membrane bound particles
FITC-Labeled Bacteria
• The remaining fluorescence
represents internal particles

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 Ionic Flux Determinations
■ Calcium Indo-1
■ Intracellular pH BCECF
How the assay works:
• Fluorescent probes such as Indo-1 are able to bind to
calcium in a ratiometric manner
• The emission wavelength decreases as the probe binds
available calcium
0.8

Ratio: intensity of 460nm / 405 nm signals

1000

0.7

0.6
800

0.5
RATIO [short/long]

600

0.4

0.3
400

0.2
200

0.1
Stimulation
Time
0

0 36 72 108 144 180 0

Time (Seconds) 0 50 (seconds)
100 150 200

Flow Cytometry Image Analysis

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 Research Applications
■ Phenotypic Analysis:
– Immunophenotypic Analysis of Peripheral Blood Lymphocytes
– Detection of Cytokine Receptors
– Enumeration of CD34+ Hematopoietic Stem and Progenitor Cells
– Measurement of CD40 Ligand Expression on Resting and In Vitro-Activated T Cells
■ Nucleic Acid Analysis:
– Analysis of DNA Content and DNA Strand Breaks for Detection of Apoptotic Cells
– DNA Content Measurement for DNA Ploidy and Cell Cycle Analysis
– Analysis of DNA Content and BrdU Incorporation
■ Cell Function:
– Oxidative Metabolism of Neutrophils
– Measurement of Intracellular pH
– Analysis of Mitochondrial Membrane Potential
– Reporters of Gene Expression
– Measurement of Intracellular Calcium Ions
– Intracellular Cytokines
■ Microbiological Applications:
– Antibiotic Susceptibility
– Cell Cycle Analysis of Yeasts
– DNA/RNA Analysis of Phytoplankton
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 Clinical Applications
■ Cancer therapy monitoring:
– DNA content of tumor cells is determined to assess the prognosis of cancer patients.
– DNA specific probes bind directly to the DNA to enable evaluation of normal and abnormal
cells.
– The measurement of DNA content in cells was one of the earliest applications of flow
cytometry.
– DNA specific dyes stain cells stoichiometricly, this means that the amount of stain is directly
proportional to the amount of DNA.

■ Cell function analysis:

– Neutrophils (polymorphonuclear leukocytes) are a major contributor to the early
inflammatory response and are a primary source of toxic oxygen metabolism.
– The function of these cells is important in combating bacterial infections.
– Flow cytometry has been used to study many disorders of the neutrophil.

■ Many platelet defects responsible for bleeding disorders are diagnosed using flow cytometry to
identify glycoprotein receptors.
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 Applications
■ Chromosome Karotyping
– Flow cytogenetics is the classification and purification of
chromosomes.
– Human as well as other animal chromosomes have been isolated and
genetic libraries constructed.
– All chromosomes can be identified and sorted by using two fluorescent
probes, Hoechst 33258 and Chromomycin A3.

■ Fetal Cell Detection

– During the first trimester fetal cells cross the placenta and can be
isolated from maternal blood. Fluorescence In-Situ Hybridization (FISH)
uses chromosome-specific DNA probes that can identify any
chromosomal abnormalities that may be present in the fetus. This
procedure may replace amniocentesis as a noninvasive method of
determining fetal status.
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 Applications
■ Minimum Residual Disease
– Patients that appear to be in complete remission can be found to have residual tumors cells that
are too few in number to be counted by standard techniques.
– Flow cytometry can detect these rare cells before they proliferate and cause the patient to
relapse.
■ Flow Cytometric Crossmatch (FCXM)
– assess potential solid organ allograph recipients.
– Circulating alloantibodies at levels too low to be detected by standard methods can be detected
by a flow cytometric crossmatch (FCXM).
– transplants done based on a negative FCXM are more successful.
■ Transplantation
– Patients with neoplastic disease require a minimum number of 2-5 X 106/kg recipient body
weight of CD34+ cells for engraftment. Flow cytometry and the identification of the CD34 antigen
on hematopietic progenitor cells has made this possible.
– Patients with type 1 diabetes may have pancreatic islets transplanted thus eliminating the need
for daily injections of insulin.

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 Commercial Applications

■ Biology and Cytometry of Sperm Sorting

■ Spermatozoal Differences
– Sex pre-selection is based on identifying differences between X- and Y-
bearing sperm
– The X chromosome contains about 4% more DNA in cattle and horses
than the Y chromosome.
– This difference in DNA content can be used to distinguish and select X
from Y bearing sperm.

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