FLOW CYTOMETRY

Flow cytometry is a sophisticated instrument measuring multiple physical characteristics of a

single cell such as size and granularity simultaneously as the cell flows in suspension through
a measuring device. 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. This
approach makes flow cytometry a powerful tool for detailed analysis of complex populations
in a short period of time.

PRINCIPLE: The basic principle of flow cytometry is based on the measurement of light
scattered by particles, and the fluorescence observed when these particles are passed in a
stream through a laser beam. It involves the passage of cells in single file in front of a laser so
they can be detected, counted and sorted. Cell components are fluorescently labelled and
then excited by the laser to emit light at varying wavelengths.

FIGURE: Schematic of a common flow cytometer, illustrating the fluidic, optical, and
electronic systems.
• Forward-scattered light (FSC) is proportional to the cell-surface area or size of the cell.
It is a measurement of mostly diffracted light and detects rays that are just off the
axis of the incident laser beam dispersed in the forward direction by a photodiode.
• Side-scattered light (SSC) indicates the cell granularity or internal complexity of the
cells. SSC is a measurement of mostly refracted and reflected light that occurs at any
interface within the cell where there is a change in the refractive index.
• The measurements of FSC and SSC are used for the differentiation of cell types in a
heterogeneous cell population.
• In a mixed population of cells, different fluorochromes can be used to distinguish
separate subpopulations.
 • The fluorescence pattern of each subpopulation, combined with FSC and SSC data, can
be used to identify which cells are present in a sample and to count their relative
percentages.
• The electronics system then converts the detected light signals into electronic signals
that can be processed by the computer.

Protocol/Procedure/Process/Steps of Flow Cytometry

The process of flow cytometry consists of the following:
Sample Preparation
• Before running in the flow cytometers, the cells under analysis must be in a single-cell
suspension.
• Clumped cultured cells or cells present in solid organs should first be converted into a
single cell suspension before the analysis by using enzymatic digestion or
mechanical dissociation of the tissue, respectively.
• It is then followed by mechanical filtration should to avoid unwanted instrument clogs
and obtain higher quality flow data.
• The resulting cells are then incubated in test tubes or microtiter plates with unlabeled
or fluorescently conjugated antibodies and analyzed through the flow cytometer
machine.

Antibody Staining
• Once the sample is prepared, the cells are coated with fluorochrome-conjugated
antibodies specific for the surface markers present on different cells. This can be
done either by direct, indirect, or intracellular staining.
• Indirect staining, cells are incubated with an antibody directly conjugated to a
fluorophore.
• In indirect staining, the fluorophore-conjugated secondary antibody detects the
primary antibody
• The intracellular staining procedure allows direct measurement of antigens presents
inside the cell cytoplasm or nucleus. For this, the cells are first made permeable and
then are stained with antibodies in the permeabilization buffer.

Running Samples
• At first, control samples are run to adjust the voltages in the detectors.
• The flow rates in the cytometer are set and the sample is run.
Applications/Uses
Flow Cytometry is used in several fields including molecular biology, pathology, immunology,
virology, plant biology, and marine biology. Some of the common application include:
• It is used in clinical labs for the detection of malignancy in bodily fluids like leukemia.
• Cytometers like cell sorters can be used to separate the cells of interest in separate
collection tubes physically.
• It can be used for the detection of the content of DNA by using fluorescent markers.
• Flow cytometers allow the analysis of replication cells by using fluorescent dye for four
different stages of the cell cycle.
• Acoustic flow cytometers are used in the study of multi-drug resistant bacteria in the
blood and other samples.
• The different stages of cell death, apoptosis, and necrosis can be detected by flow
cytometers based on the differences in the morphological and biochemical changes.
 Fluorescence-activated Cell Sorting (FACS)
Fluorescence-activated cell sorting (FACS) is a specialized type of flow cytometry. It provides
a method for sorting a heterogeneous mixture of biological cells into two or more containers,
one cell at a time, based upon the specific light scattering and fluorescent characteristics of
each cell. It is a useful scientific instrument, as it provides fast, objective and quantitative
recording of fluorescent signals from individual cells as well as physical separation of cells of
particularinterest.

The cell suspension is entrained in the center of a narrow, rapidly flowing stream of liquid.
The flow is arranged so that there is a large separation between cells relative to their
diameter. A vibrating mechanism causes the stream of cells to break into individual droplets.
The system is adjusted so that there is a low probability of more than one cell per droplet.
Just before the stream breaks into droplets, the flow passes through a fluorescence
measuring station where the fluorescent character of interest of each cell is measured. An
electrical charging ring is placed just at the point where the stream breaks into droplets. A
charge is placed on the ring based on the immediately prior fluorescence intensity
measurement, and the opposite charge is trapped on the droplet as it breaks from the stream.
The charged droplets then fall through an electrostatic deflection system that diverts droplets
into containers based upon their charge. In some systems, the charge is applied directly to
the stream, and the droplet breaking off retains charge of the same sign as the stream. The
stream is then returned to neutral after the droplet breaks off.
Fluorescence-activated ce

ll sorting (FACS)

A fluorescence-activated cell sorter (FACS)

An antibody specific for a particular cell surface protein is associated to a fluorescent molecule
and then added to a mixture of cells. For fluorescence when the specific cells pass through a
laser beam they are monitored. Droplets containing single cells are given a positive or
negative charge, based on whether the cell has limited the fluorescently-tagged antibody or
not. Droplets containing a single cell are then detected by an electric field into collection tubes
according to their charge.

Interests are first labeled with an antibody which is individual for a particular cell surface
molecule. Antibody is coupled to a fluorescent dye, like when in a narrow stream the
individual cells pass a laser beam in single file, the fluorescence of each cell is measured. A
vibrating nozzle then forms small droplets which each containing a single cell which are given
a negative or positive charge based on whether the cell they contain is fluorescing. A strong
electric field defects the various charged droplets into separate containers so that each
container has a homogeneous population of cells eventually with respect to the cell surface
molecule tagged along fluorescent antibody. For biochemical analysis or grown in culture
these homogeneous populations may then be used. By flow cytometry the RNA and DNA
content of a cell can be measured also.

Go through the below link depicting video on Flow Cytometry

https://youtu.be/l4MBW9R1i2U-
https://youtu.be/EQXPJ7eeesQ
https://youtu.be/vibrlg_RZA0 - PART- I

Go through the below link depicting video on FACS

https://youtu.be/7bC Zx5xPwt0
https://youtu.be/6KyTJhWzCkk- PART - II