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