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Analysis of Apoptosis by Flow Cytometry (CAT#: STEM-CBT-0060-WXH)

Introduction

Apoptosis, or programed cell death, is a phenomenon that is frequently examined in immunology and other fields of study. It is used by the immune system to maintain the homeostasis by removing cells without triggering an inflammatory response. This is in contrast to necrosis, a type of cell death that does trigger an inflammatory response. Apoptosis is the mechanism of cell death for clonally expanded T cells following an immune response, for self-targeting T cells, for autoreactive B cells, and multiple other cells in the immune system.
Flow cytometry is widely used to analyze apoptosis due to the versatility afforded by its several technological benefits:
Multiparameter analytical capabilities – Ability to analyze several parameters of apoptosis in a single assay
High-throughput analysis of large numbers of cells
Quantitative data gathering on percentages, as well as absolute number of cells
Single cell resolution – individual cell-based mode of analysis




Principle

Expression of proteins involved in apoptosis
Activation of caspases
Changes in the mitochondrial membrane potential
Changes in the plasma membrane
Cell shrinkage
Chromatin changes
DNA degradation

Applications

• Conclusive identification of phases of apoptosis.
• Multiparameter apoptosis detection.
• Reproducible apoptotic protein analysis using recombinant antibodies.

Procedure

1.Collect cells
2.Wash cells
3.Add staining solution and incubate
4.Examine by flow cytometry

Materials

Annexin V
Antibodies specific to death receptors
Collapse of mitochondrial membrane potential: JC-1, TMRE, and TMRM
Detect cleaved nuclear DNA: trypan blue,Hoechst 33342

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