Question (75795) | Kinetic Apoptosis Kit (Microscopy) (ab129817)

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Question

I am a PhD student having trouble finding something to measure apoptosi/necrosis in real-time.
I'd like to look at two cell types in direct co-culture and detect early and late apoptosis and necrosis in one of the cell types using real-time fluorescence microscopy.
I will probably end up doing flow cytometry, as it looks to be the most accurate way of differentiating a mixture of cells, but it would be nice to do some live-cell fluorescence imaging over several days to watch how the process unfolds.
Most apoptosis/necrosis detection assays are end-point assays and require you to stain the target cells after the induction of apoptosis. Is there anything that you can pre-stain the cells with so that you can put them in direct co-culture with another cell type that is killing them and monitor over time (say at least 3 days) for apoptosis and necrosis in the stained population alone?

Answer

The product that is best suited to what you want to do is ab129817 for microscopy. We also have ab129816, the same reagents adapted for flow cytometry. These detect exposed phosphatidylserine. However, this occurs as an intermediate step during apoptosis, not early or late. Detection of early and late events by flow cytometry are discussed in the following two references but I am not sure if these approaches can be adapted to real-time detection via microscopy over several days.

Exp Cell Res. 2002 Jul 1;277(1):1-14. http://www.ncbi.nlm.nih.gov/pubmed/12061813

Cytometry. 1996 Jun 1;24(2):106-15. http://www.ncbi.nlm.nih.gov/pubmed/8725659

I am also not sure how easily late-stage apoptosis can be distinguished from necrosis.

For more information about the principle of the ab129816 and ab128817 assays, we recommend the following reference.

Kim YE et al. Monitoring apoptosis and neuronal degeneration by real-time detection of phosphatidylserine externalization using a polarity-sensitive indicator of viability and apoptosis. Nat Protoc 5:1396-405 (2010).

http://www.ncbi.nlm.nih.gov/pubmed/20671723?dopt=Abstract

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