We are thinking of using annexin-V and PI kit to asses the necroptotic cells through Flowcytometry. Please suggest if there are specific FACS kit available for the same.
Thank you
Dear Firdos,
Annexin V and PI could work (but with some tweaking), in the sense that it will differentiate between healthy cells (- Annexin, - PI), early apoptotic (+/-) and late apoptotic/necrotic/necroptotic (-/-).
Now, the double negative one is not so informative given that it lumps three kinds of cell death into one. Here you can use necrostatin to specifically halt necroptotic death.
Another idea would be to utilize immunofluorescent targeting of key proteins in necroptotic pathway (RIPK3, RIPK1, and MLKL, the latter in its phosphorylated form) so you can detect the population with higher expression using flow cytometry.
Check also these links:
http://www.bioradiations.com/assessing-cell-health-necroptosis/
https://www.ncbi.nlm.nih.gov/m/pubmed/29175336/
I wish you the best!
GFP-Certified Apoptosis/Necrosis detection kit, (ENZ 51002), Enzo Life Sciences
Hi Firdos,
Yes the best way is to use my new assay published in Methods Feb 2018 using a fixable live/dead stain then fix and perm and use RIP3-PE (Santa Cruz and Caspase-3-BV650 (BD). Live Necroptotic cells have the phenotpye RIP3High+ve/Caspase-3-ve, early apoptosis RIP3-ve/Caspase-3+ve while Double Positives indicate RIP1-dependent apoptosis - you can do this on live and dead cells, see the publication HL Lee, R Pike, MHA Chong, A Vossenkamper, G Warnes. Simultaneous flow cytometric immunophenotyping of necroptosis, apoptosis and RIP1 dependent apoptosis Methods, 1, 134-135, 56-66, 2018.
Regards
Gary
@Firdos Ahmad, Erratum on my previous response, the text should read:
" and late apoptotic/necrotic/necroptotic (+/+).
Now, the double positive one is not so informative..."
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