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Flow Cytometry apoptosis detection

two key reagents for apoptosis analysis...

Biostatus offer two flow cytometry approaches to the study of apoptosis:-

1. Discriminate live, dead and apoptotic cells using DRAQ7™

2. See apoptotic populations using sub-G1 cell cycle analysis with DRAQ5™

 

1. Tracking apoptosis - discriminating intact, leaky & apoptotic cells

See the example protocol for use of DRAQ7™ in flow cytometry. Navigate to the main product section on DRAQ7™ for further information.

DRAQ7™ enters and labels ONLY membrane-compromised cells.

The flow cytometric data  below shows apoptosis (compromised membranes permitting entry of DRAQ7) of human Jurkat cells exposed to 0.1 - 2.0 µM staurosporine for 24h compared to negative controls.

DRAQ7 FCM Apop StaurosporineDRAQ7™ can be used in flow cytometric analysis to mark membrane-compromised cells, in combined with reagents such as Annexin V (see example), mitochondrial membrane probes and other reporters of cell health.

DRAQ7™ can be used in apoptosis assays, cytotoxicity assays, cytolytic antibody testing, cell health assays. 

Due to its far-red emission signal, DRAQ7™ can be easily combined with multiple other fluorophores; up to 4 using the single 488 nm excitation source (argon-ion laser).  It avoids unwanted signal spillover into the useful visible spectrum (e.g. propidium iodide's overlap with R-PE, JC-1 (see example) and similar "orange" emitting fluorophores).

2. Sub-G1 cell cycle analysis as a marker of apoptotic DNA fragmentation

With DRAQ5™ you can generate cell cycle data and measure the presence of a sub-G1 fraction. This indicates DNA fragmentation resulting from apoptosis. The figure below shows the loss of small, sub-G1 DNA fragments when using a DNA dye (e.g. ethidium bromide) that requires permeabilization compared to DRAQ5 which, as a live cell permeant dye, does not. DRAQ5 subG1 detection

Snyder et al (2004) used this strategy when studying the potential for a peptide treatment of advanced peritoneal carcinomatosis. Cultured Namalwa human B-lymphoma cells were directly stained with DRAQ5™ and analysed by flow cytometry.

Due to the far-red emission of DRAQ5 DNA staining could also be preceded by a immunochemical staining for antigens to determine phenotype of apoptotic cells in primary / heterogeneous samples.  See cell cycle analysis for more information on this topic.  See the DRAQ5 protocol.