Flowcytometry
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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.
Flowcytometry
- 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 )