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Flow cytometry
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Flow cytometry

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  1. Flow cytometry

  2. Powerful technique for analysis of multiple parameters of individual cells within heterogeneous populations multiple physical characteristics of single particles or cells What is flow cytometry?

  3. scheme of flow cytometry

  4. The fluidic system & optics system • heart of the system • where the laser and the sample intersect and the optics collect the resulting scatter and fluorescence

  5. The fluidic system • Particles or cells are passed through the laser beam one at a time Laser Hydrodynamic focusing

  6. Size comparison by flow cytometry • ~ 1 ~ 15 uM size detection

  7. Forward scatter • Light is quantified by a detector that converts intensity into voltage • Cell size

  8. Side scatter • Granularity and structural complexity inside the cell

  9. Scatter plot SSC FSC

  10. Energy state diagram

  11. Using fluorescence in flow cytometry

  12. Fluorescence detection

  13. Two color experiment

  14. Two color experiment -/+ +/+ -/- +/-

  15. Compensation

  16. Compensation • No-stained sample • Only FITC stained sample • Only PE stained sample uncompensated compensated PE FITC

  17. Application (apoptosis) • Apoptosis (FSC/SSC) Apoptosis SSC Necrosis FSC

  18. Application (apoptosis) • Apoptosis (Annexin V/PI assay) Normal Cell Apoptotic Cell : Phosphatidyl Serine : Annexin V-FITC

  19. Application (apoptosis) • Apoptosis (Annexin V/PI assay) Control Anti-Fas Ab Propidium Iodide (PI) Annexin V-FITC

  20. Application (cell cycle) Apoptotic DNA Histogram A typical DNA Histogram G0-G1 G2-M S Apoptotic cells Normal G0/G1 cells # of Events Fluorescence Intensity