PG AND RESEARCH DEPARTMAENT OF MICROBIOLOGY,SRI PARAMAKALYANI, COLLEGE,ALWARKURICHI - 627 412

1. Reaacredited with B grade with a CGPA of 2.71 in the second cycle of NAAC
affiliated to manomanium sundaranar university, tirunelveli.
Post graduate & Research Centre – Department of Microbiology
(government aided)
ACADEMIC YEAR 2021-2022
II SEM CORE: IMMUNOLOGY
UNIT-1
FLOW CYTOMETRY
T.RAMAR. SUBMITTED TO
REG NO:20211232516121 GUIDE:DR.S.VISWANATHAN
I M.SC MICROBIOLOGY ASSISTANT PROFFESSOR&HEAD
ASSIGNED ON: 08/12/2021 SPKC - ALWARKURUCHI

2. Indroduction
• Flow cytometry is a popular laser – based
technology to analyze the characteristics of cells or
particles.
• It is the measurement (metry) of cells (cyto) in a fluid
(flow).
• With flow cytometry, we detect and measure the
physical, chemical and fluorescent characteristics of
cells.

3. • Cell size
• Cell complexity
• Cell structure
Characteristics

4. Uses
• Cell sorting
• Cell counting
• Protein Expression
• Cell viability

5. Principle
• Flow cytometer is composed of three mani components:
• The Flow system (fluidics)
cells in suspension are brought in single file past.
• The optical system (light sensing) .
a focused laser which scatter light and emit fluorescence that is
filtered and collected.
• The Electronic system (signal processing)
emitted light is converted to digitized values that are stored digitized
values that are stored in a file for analysis.

6. Flow cytometry
•Fluidics : transport the sample to laser beam.
•Optics system : lasers and lenses.
•Electronic system : Amplifies signals, converts
signal, into Graphs.

7. Hydrodynamic Focusing
• Sample is injected.
• Sheath fluid helps to carry and
align cells.
• It pass cell one by one in a narrow
channel.
• This results in laser to focus one
cell at a time.

8. Scattering of Light
Two tybes:
1. Forward scattering : size of the
cell.
2. Side scattering : complexity of
the cell.
components present in the cell.

9. 2D Histogram
• If the Scattering is high.
• Peak of the graph is
high.
• Size and Complexity of
the cells is high.

10. Fluorescence Activated Cell Sorting (FACS)
• A tybe Flow cytometry
Technique used in
sorting (separating)
cells Based on the
Fluorescence material
attached to it.

11. Sample Preparation for FACS
• The clustered or clumped cells are Suspended
into single cells.
• They are treated with different Fluorochromes
specific to specific molecules (proteins).
• Antibodies conjugated with Dyes can also be
used for Specific antigens on the cells.
• The fluorescent material excite at certain
wavelength of light.
• And emit signals which is detected by
detectors.

12. Mechanism
• The cells flow one by one due to Hydrodynamic
Focusing.
• Analysed based on ,
• Scattering of light – size & complexity of cells.
• Fluorescence – tybe of the cell (protein
Expression)
• The cells enter a Drop formation and Electric
charging Ring.
• It provides a vibrating mechanism.
• Based on the size, complexity and tybe, charge is
imparted based on the cells.
• The cells are separated based on their acquired
charge using deflector plates in separate
containers.

13. Application
• Molecular biology.
• Immunophenotyping.
• Cell Biology.
• Haematology.
• Cell counting.
• Pharmacology.

14. Reference
• https://w.w.w.ncb.nlm.nih.gov/PMC
/articles/pm/5939936/
• https://w.w.w.bosterbio.com
• https://w.w.w.academic.oup.com