1. Cell viability studies Sepideh Khoshnevis

2. The Goal To distinguish live cells from dead and apoptotic cells in order to calculate the the percentage of viable cells for each experiment

3. Cell viability study Trypan blue studies –Finding data for a small population –a good first order approximation Flow cytometry –Processing a large volume of data in a reasonable time

5. BD FACSCalibur

6. How does flow cytometer work? It is made up of three systems –Fluidics –Optics Laser Filters –electronics

7. introduction to flow cytometry, BD biosciences

8. fluidics Hydrodynamic focusing introduction to flow cytometry, BD biosciences

9. Light Scatter Forward scatter (FSC) –Proportional to cell size Side scatter (SSC) –Proportional to cell granularity or internal complexity FSC vs. SSC plot –Isolating mixture of cell population

10. Differentiation of cell types introduction to flow cytometry, BD biosciences

11. Fluorescence Fluorescent dyes Fluorochrome conjugated antibodies

12. Optical bench diagram introduction to flow cytometry, BD biosciences

13. Signal detection When the particles enters the laser beam light signal is being produced Light is converted to voltage by photodetectors –Photodiode –Photomultiplier tube Size of voltage pulse depends on the number of photons detected The height of the voltage pulse dictate what channel it falls under

14. Creation of voltage pulse

16. Data display Histogram Dot plots Multi dimensional plots

17. Current protocols in cytometry

20. Gating Isolating the population of interest –Cluster analysis Further analysis of a subpopulation