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Analysis of Cell Viability by Flow Cytometry (CAT#: STEM-CBT-0057-WXH)

Introduction

Cell viability can be measured to either exclude dead cells from analysis or to study the physiological state of cells. In both cases, fluorescent or non fluorescent dyes are available to distinguish dead and live cells.<br />Dead cells tend to non specifically bind many reagents. When immunophenotyping a cell population it is always useful to remove dead cells from acquired data to avoid false positive result.<br />Cell viability can be measured by changes in morphology or by changes in membrane permeability. Changes in morphology can be detected by forward and side scattered characteristics but this can be a crude measurement of viability. But, a more accurate way of measuring cell viability is by making use of cell exclusion of certain dyes or cell uptake and retention of others.




Principle

1. Dye exclusion: Live cells have membranes that are still intact and exclude a variety of dyes that easily penetrate the damaged, permeable membranes of non-viable cells. (Propidium iodide, DAPI)
2. By the binding of a dye to amines within a cell to determine if the cell membrane is intact.

Applications

• Evaluating a cells response to drug treatments or other environmental factors.
• Distinguish dead cells in a cell suspension in order to exclude them from analysis.
• Measuring the results of cell proliferation.
• Testing for cytotoxic effects of compounds.

Procedure

1.Harvest and wash cells
2.Resuspend cells
3.Add staining solution to a control tube of unstained cells and incubate for a while, adjust flow cytometer settings
4.Add staining solution prior to analysis
5.Determine fluorescence with a FCM instrument

Materials

Dye exclusion: Propidium iodide, DAPI
Amine binding dyes: the Live/Dead reagents, Zombie dyes or Fixable Viablity dyes

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