Cell viability studies
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Transcript of Cell viability studies
Cell viability studies
Sepideh Khoshnevis
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
BD FACSCalibur
How does flow cytometer work?
• It is made up of three systems– Fluidics– Optics
• Laser• Filters
– electronics
introduction to flow cytometry, BD biosciences
fluidics
• Hydrodynamic focusing
introduction to flow cytometry, BD biosciences
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
Differentiation of cell types
introduction to flow cytometry, BD biosciences
Fluorescence
• Fluorescent dyes• Fluorochrome conjugated antibodies
Optical bench diagram
introduction to flow cytometry, BD biosciences
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
Creation of voltage pulse
Data display
• Histogram• Dot plots• Multi dimensional plots
Current protocols in cytometry
Current protocols in cytometry
Current protocols in cytometry
Gating
• Isolating the population of interest– Cluster analysis
• Further analysis of a subpopulation