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

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