Hi everyone,
I am trying to perform the FLICA apoptosis assay with my MSCs. However, although the cells look healthy upon inspection and the viability is greater than 90% with cell counting, the FLICA assay shows that all the cells are apoptotic. I have tried using both trypsin and accutase when harvesting the cells.
It would really be helpful if anyone could give me an idea with regards to what could be going wrong, or anything else I could try?
Thank you in advance.
Dear Nishant!
You look at this article, please
Article Fluorochrome-Labeled Inhibitors of Caspases: Convenient In V...
FLICA containing VDVAD, DEVD, VEID, YVAD, LETD, LEHD, and AEVD peptide moieties were expected to preferentially bind to activated caspases-2, -3, -6, -1, -8, -9, and -10, respectively.
The difference that you observe in relation to cell death when using the FLICA analysis and cell morphology is most likely due to the fact that during the FLICA analysis, caspases are activated, which are markers of early aptopotic death (2-3 hours), and morphological changes during apoptosis are manifested in later stages (in a day or more). Therefore, your cells still look healthy.
You can additionally perform other tests of propidium iodide / annexin-5 to verify cell death.
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