This document provides an overview of flow cytometry. [1] Flow cytometry works by measuring the physical and chemical characteristics of particles like cells as they flow through a laser beam. [2] It uses optical and electronic systems to direct scattered and fluorescent light from cells to detectors. [3] This allows measurement of cell properties like size, granularity, and identification using fluorescent antibodies to distinguish cell types.

1. Demystifying Flow Cytometry
Presented by
Dr D Sumanth Kumar
Moderators
Prof Dr Kumuda
Dr Devendar Reddy

2. Basic Principle

Measuring and analyzing physical &/
chemical characteristics of particles (cells)
as they flow through a beam of light
( Laser)

0.2 to 150 μm

Sample: Blood/Bone marrow; Anti-
coagulated di-Na EDTA/ Na citrate/ Heparin

Solid Tissue; RPMI (Roswell Park Memorial
Institute) medium & minced

3. Flow Cytometer

The Flow System

The Optical System

Electronic System

7. Flow Cytometer: Flow System

Stream of Sheath fluid (0.9% Saline / PBS)

Hydrodynamic focusing

Sample pressure Vs Sheath fluid pressure

10. Flow Cytometer:The Optical System

Laser illumination (M/C Argon Laser 488nm)

Optical mirrors and filters : direct scattered
light and fluorescent light to appropriate
photo detectors

Light scatter: 1) Forward light scatter;
surface area 2)Side scatter; cytoplasmic
granularity & nuclear complexity

Light scatter example Lymphocytes vs
Neutrophils ..Continued

14. Flow Cytometer:The Optical System
….

Flourescence:Monoclonal antibody;
Principal; direct & indirect staining

Fluorescent dyes/ Antibodies conjugated
with Fluorochromes

FITC (Flouroscene iso-thio-cyanate); green;
510-560 nm ( ~525 nm)

PE (Phyco-erythrin); red; ~570 nm

17. Flow Cytometer: Electronic system

Photodetectors & ADC ( Analog to digital
converter)

PMT ( Photomultiplyer tubes)

Photodiode

Log amplyfier & Linear amplyfier

ADC: Organize signals to form data plots
(histograms, dot plots, 3D histograms)
….continued

23. Flow Cytometer: Electronic system
…..

Setting up a Threshould

Gating

24. USES

Cell Markers: Monoclonal antibody
technology; CD markers

DNA content: Laser with significant UV
output and florescent DNA stains
1) Propidium iodide 2)HOECHEST 33342/
DAPI ( di-amidino phenyl-indole) ; adinosine
/ thiamine 3)Mithramicyin & chromomycin
A3; guanine/ cytosine
….continued

25. USES
 Cell Cycle Analysis:

27. Disadvantages

Low Cell Throughput Rate

Requirement of Highly Trained Operators

Expensive( $100,000 to $250,000 )