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PBS 7413 FLOW CYTOMETRY LABORATORY

PBS 7413 FLOW CYTOMETRY LABORATORY. COURSE OBJECTIVES. UNDERSTAND CLINICAL AND RESEARCH APPLICATIONS OF FLOW CYTOMETRY TECHNOLOGY PERFORM STAINING TECHNIQUES ANALYZE DATA INTERPRET DATA AS PRESENTED IN BASIC SCIENCE AND CLINICAL SCIENCE LITERATURE.

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PBS 7413 FLOW CYTOMETRY LABORATORY

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  1. PBS 7413FLOW CYTOMETRY LABORATORY

  2. COURSE OBJECTIVES • UNDERSTAND CLINICAL AND RESEARCH APPLICATIONS OF FLOW CYTOMETRY TECHNOLOGY • PERFORM STAINING TECHNIQUES • ANALYZE DATA • INTERPRET DATA AS PRESENTED IN BASIC SCIENCE AND CLINICAL SCIENCE LITERATURE

  3. Flow Cytometry Principles And Applicationswww.vetmed.lsu.edu/facs

  4. FLOW CYTOMETRY Fluorescence activated cell sorting (FACS) is a technology that allows simultaneous measurement of multiple physical and chemical characteristics of a single cell. These measurements are made on a per cell basis at routine rates of 500 to 70,000 cells per second in a moving stream.

  5. FACS COMPONENTS • LASER • OPTICS • FLUIDICS • ELECTRONICS

  6. LASER • ARGON • EMISSION LINE OF 488 nm • VISIBLE LIGHT

  7. OPTICS • BEAM SPLITTERS • DICHROIC MIRRORS • LONG / SHORT PASS FILTERS • BAND PASS FILTERS

  8. FLUIDICS • SAMPLE FLUID • SHEATH FLUID • FLOW CELL • NOZZLES, 70, 100, 400 um

  9. ELECTRONICS • PHOTOMULTIPLIER TUBES (PMTs) • PHOTODIODE • MACINTOSH G3 COMPUTER

  10. DATA PARAMETERS • FORWARD LIGHT SCATTER • 90o OR SIDE SCATTER • FLUORESCENCE EMISSION

  11. FORWARD SCATTER • LIGHT SCATTER USED IS LOW ANGLE • SENSITIVE TO CELL SIZE AND SURFACE AREA • LIVE / DEAD DISCRIMINATION

  12. SIDE SCATTER • LIGHT SCATTER USED IS 90o • SENSITIVE TO INTERNAL STRUCTURES

  13. RIGHT ANGLE LIGHT SCATTER FORWARD ANGLE LIGHT SCATTER

  14. BEST ANALYSIS USING FORWARD AND SIDE SCATTER TOGETHER; ANALYSIS OF HETEROGENEOUS POPULATIONS

  15. PERIPHERAL BLOOD MONONUCLEAR CELLS SSC SSC FSC FSC LYSED PERIPHERAL WHOLE BLOOD SSC SSC FSC FSC

  16. FLUORESCENCE • DETECT BINDING OF LABELED LIGAND • LIVE/DEAD DETERMINATION • DNA/RNA CONTENT

  17. FLUORESCEIN TEXAS RED PHYCOERYTHRIN ALLOPHYCOCYANIN EXCITATION OR EMISSION PROPIDIUM IODIDE RHODAMINE WAVELENGTH (NM)

  18. SINGLE BEAM EXCITATION THREE COLOR EMISSION

  19. UNCOMPENSATED CD3 RED FLUORESCENCE CD19 COMPENSATED CD3 CD19 GREEN FLUORESCENCE

  20. APPLICATIONS • IMMUNOPHENOTYPING • CELL CYCLE ANALYSIS • APOPTOSIS • CELL FUNCTION • CELL SORTING

  21. CELL SURFACE STAINING SINGLE CELL SUSPENSION MONOCLONAL ANTIBODY FLUOROCHROME CONJUGATED MONOCLONAL FLUORESCENCE ANTIBODY CONJUGATED SECOND REAGENT FACS ANALYSIS / CELL SORTING

  22. ISOTYPE CONTROL SSC FSC UNGATED R1 IgG2a PE IgG2a PE IgG1 FITC IgG1 FITC

  23. IMMUNOPHENOTYPING 16% CD4+ 55% CD8+ CD8 PE CD4 FITC

  24. ADVANTAGES OF FACS • PRECISION • SENSITIVITY • NO PHOTOBLEACHING • VOLUME • SINGLE CELL ANALYSIS • SAMPLING STATISTICS • SORT CELLS IN SEPARATE TUBES

  25. CELL CYCLE ANALYSIS • FACS IS METHOD OF CHOICE FOR FAST, ACCURATE DETERMINATION OF CELL CYCLE DISTRIBUTIONS • DETERMINATION OF ANUEPLOIDY • % OF S PHASE

  26. Normal Cell Cycle events 2N 4N DNA content

  27. TUMOR DNA STAINING SINGLE CELL SUSPENSION PERMEABILIZE WITH METHANOL DIGEST DS RNA WITH RNASE STAIN DNA WITH PROPIDIUM IODIDE

  28. Cell Cycle Analysis G0/G1 = 60% S = 13% G2/M = 27% EVENTS DNA CONTENT

  29. CELL CYCLE ANALYSIS G0/G1 G0/G1 = 79.5% S = 12.7% DIPLOID TUMOR G2/M = 7.8% DNA INDEX = 1.11 G2/M S

  30. MEASUREMENT OF APOPTOSIS • APOPTOSIS IS PROGRAMMED CELL DEATH WHERE THE CELL GOES THROUGH A HIGHLY REGULATED PROCESS OF “DYING” • CHARACTERISTICS ARE CONDENSATION OF THE CHROMATIN MATERIAL AND BLEBBING OF NUCLEAR MATERIAL • OFTEN ACCOMPANIED BY INTERNUCLEOSOMAL DEGRADATION OF DNA

  31. CYTOMETRY IN CELL NECROBIOLOGY CELL DEHYDRATION APOPTOTIC BODIES APOPTOSIS CELL AND MITOCHONDRIAL SWELLING PLASMA MEMBRANE RUPTURE NECROSIS

  32. DETECTION METHODS FOR APOPTOSIS • STRAND BREAKS IN DNA CAN BE LABELED ENZYMATICALLY IN SITU BY TdT • STRAND BREAKS IN DNA CAN BE LABELED DIRECTLY USING dUTP • MEMBRANE PHOSPHATIDYL SERINE CAN BE DETECTED WITH ANNEXIN V

  33. APOPTOSIS 31% Necrotic PROPIDIUM IODIDE 33% Viable 35% Apoptotic ANNEXIN V FITC

  34. CELL FUNCTION • STAINING OF ACTIVATION ANTIGENS • INTRACELLULAR Ca2+ LEVELS • INTRACELLULAR pH LEVELS • MEMBRANE POTENTIAL • CYTOKINE SECRETION

  35. ACTIVATED LYMPHOCYTES SSC FSC

  36. ACTIVATION ANTIGENS 33% 27% CD69 PE 7% CD25 FITC

  37. CELL SORTING AS LIQUID IS EJECTED INTO AIR, IT WILL FORM DROPLETS BY VIBRATING THE NOZZLE AT A DEFINED FREQUENCY, THE SIZE OF THESE DROPLETS AND THE POSITION ALONG THE STREAM WHERE THEY FORM CAN BE CONTROLLED WITH PRECISION. A DROPLET CONTAINING A CELL IS APPLIED EITHER A NEGATIVE OR POSITIVE CHARGE AND SORTED BY PASSING THROUGH AN ELECTRIC FIELD.

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