cell viability studies sepideh khoshnevis. the goal to distinguish live cells from dead and...
TRANSCRIPT
The Goal
• To distinguish live cells from dead and apoptotic cells in order to calculate the the percentage of viable cells for each experiment
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
How does flow cytometer work?
• It is made up of three systems– Fluidics– Optics
• Laser• Filters
– electronics
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
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
Gating
• Isolating the population of interest– Cluster analysis
• Further analysis of a subpopulation