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1. Topic- principle &
applications of flow
cytometry
Prepared by – Dinesh M Gangoda
Enrollmentno: MPL02
Class: M. Pharm (Sem-l)
Department of pharmacology
College Name: A.R college of pharmacy and GH patel
institute of pharmacy
Guided by: Dr. Poonam D. Sachdeva
Coordinator of Pharmacology
department
Qualification: B. Pharm, M. Pharm, PhD

2. Introduction:
Flow cytometry as the name suggests is a technique for
cell counting and measurement of different properties of
the cell.
Flow – The motion characteristic of fluids.
Cyto- combining from of cells or cells.
Metry- measurement(1)
Flow cytometry is a standard laser-based technology
that is used in the detection and measurement of
physical and chemical characteristics of cells or
particles in a heterogeneous fluid mixture.
This method allows the quantitative and qualitative
analysis of several properties of cell populations from
virtually any type of fresh unfixed tissueor body fluid.
The properties measured include a particles relative
size, relative fluorescence intensity.
The use of flow cytometry has increased over the
years as it provides a rapid analysis of multiple
characteristics (both qualitative and quantitative) of
the cells.
The properties that can be measured by this process
include a particle’s size, granularity or internal
complexity, and fluorescence intensity.

3. These characteristics aredetermined usingan optical-
to-electronic coupling system that detects the cells
based on laser scattered by the cells.
A flow cytometer, despite its name, does not
necessarily deal with cells; it deals with cells quite
often, but it can also deal with chromosomes or
molecules or many other particles that can be
suspended in a fluid.(2)
Most commonly analyzed materials are:
Blood
Bone marrow aspirate
Figure: Flow cytometry instrument

4. Principle
The basicprinciple of flow cytometry is thepassageof 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.

5. Flow Cytometry – Working Principle
The basic working principle of Flow Cytometry is – this
analytical technique requires fluorescent labeling of cells,
followed by the passageof these cells suspendedin a fluid
sheet as a single file to a laser beam. The scattered and
fluorescent wavelength are detected and analyzed or
used for sorting of cells.

6. A flow cytometer has main three components:
1) fluidics
2) optics system and
3) Electronic system.
Fluidics
The purpose of the fluidics system is to transport particles
in a fluid stream to the laser beam. To accomplish this, the

7. sample is injected into a stream of sheath fluid (usually a
buffered saline solution) within the flow chamber.
The design of the flow chamber allows the sample core to
be focused in the center of the sheath fluid where the
laser beam then interacts with the particles.
Focusing is achieved by injecting the sample suspension
into the center of a sheath liquid stream. The flow of the
sheath fluid moves the particles and restricts them to the
center of the sample core.
Optics System

8. The optical system of the cytometer consists of excitation
optics and collection optics.
The excitation optics consists of the laser and lenses that
are used to shape and focus the laser beam to the flow of
the sample.
The collection optics consist of a collection lens to collect
light emitted after the particle interacts with the laser
beam and a system of optical mirrors that divert the
specified wavelengths of the collected light to designated
optical detectors.
After a cell or particle passes through the laser light, the
rays emitted on the side and the fluorescence signals are
directed to the photomultiplier tubes (PMTs), and a
photodiode collects the signals.
To achieve the specificity of a detector for a particular
fluorescent dye, a filter is placed in front of the tubes,
which allows only a narrow range of wavelengths to reach
the detector.
Electronics system
The electronic system converts the signals from the
detectors into digital signals that can be read by a
computer.

9. Once the light signals strike one side of the PMT or the
photodiode, they are converted into a relative number of
electrons that are multiplied to create a more significant
electrical current.
The electrical current moves to the amplifier and is
converted to a voltage pulse.
The highest point of the pulse is achieved when the
particle strikes the center of the beam, in which case the
maximum amount of scatter or fluorescence is achieved.
The Analog-to-Digital Converter (ADC) then converts the
pulse to a digital number.
Data analysis

11. Applications of Flow Cytometry
1.Immunophenotyping
The most common application performed on the
cytometer is immunophenotyping. This technique

12. identifies and quantifies populations of cells in a
heterogeneous sample – usually blood, bone marrow or
lymph. These cell subsets are measured by labeling
population-specific proteins with a fluorescent tag on the
cell surface. In clinical labs, immunophenotyping is useful
in diagnosing hematological malignancies such as
lymphomas and leukemia.
2.Cell Sorting
The cell sorter is a specialized flow cytometer with the
ability to physically isolate cells of interest into separate
collection tubes. The cytometer interrogates and
characterizes each cell as it passes through the laser. The
sorter then uses sophisticated electronics and fluidics to
identify and “kick” the cells of interest out of the fluidic
stream into a test tube.
3. Cycle Analysis
Flow cytometry can analyze replication states using
fluorescent dyes to measure the four distinct phases of
the cell cycle. Along with determining cell cycle replication

13. states, the assay can measure cell aneuploidy associated
with chromosomal abnormalities.
4.Apoptosis
Apoptosis, or programmed cell death, is a normal part of
the life cycle of eukaryotic cells. Cells die for a variety of
reasons:through necrosis, brought on byexternal physical
and chemical changes to the cell or through apoptosis, a
process in which cells initiate a “suicide” program through
internally controlled factors. These two distinct types of
cell death, apoptosis and necrosis, can be distinguishedby
flow cytometry on the basis of differences in
morphological, biochemical and molecular changes
occurring in the dying cells.
5.Cell Proliferation Assays
Cell proliferation assays are widely used in cell biology to
measure cellular metabolic activity in response to stimuli
such as growth factors, cytokines and other media
components. The flow cytometer can measure
proliferation by labeling resting cells with acell membrane
fluorescent dye, carboxy fluorescein succinimidyl ester

14. (CFSE). When the cells are activated, they begin to
proliferate and undergo mitosis. As the cells divide, half of
the original dye is passed on to each daughter cell. By
measuring the reduction of the fluorescence signal,
researchers can calculate cellular activation and
proliferation.
6.Intracellular Calcium Flux
Cells interact with one another and their environment
through signal transduction pathways. When these
pathways are activated, membrane-bound calcium ion
channels pump calcium into the cell and rapidly increase
the intracellular calcium concentration. The higher
calcium levels provide energy to the cell to respond to the
external stimuli. The cytometer can monitor the flux of
calcium into the cell andmeasure the extent to which cells
respond to the stimuli.
Reference:

15.  https://www.iitk.ac.in<pdf/resources/flow-
cytometry
https://microbenotes.com/flow-cytometry
https://www.slideshare.Net/JuhiArora/flowcytometr
yprinciplesandapplications- /65437818
https://www.biotecnika.org/2019/07/flow-
cytometry working- principle- applications of
https://www.seattlechildrens.org/research/resource
s/flow- cytometry/applications-of-flow-cytometry
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