I treated cells with some inhibitors and find them morphologically unhappy after 24 hours. I did Resazurin vaibility assay to measure their viability and find almost 70% reduction. To understand their death pattern I used capase 3/7 glo assay. I treated the cells for 24 hour and use caspase glo to measure caspase 3/7 acitivity and find 4 fold induction. Now my confusion is, as most of the cells are dead, is this signal because of caspases (as far I know they are not very stable for long) or there might be something else which can contribute to such signal. How stable these caspases are, are they still active when the cells are completely dead. Thanks.
? You used inhibitors of ? Caspase?
Caspase-Gl0 3/7 is a LYTIC assay meaning your cells have to be lysed in order to access Caspase.
The addition of Caspase-Glo 3/7 Reagent results in cell lysis, followed by caspase cleavage of the substrate and generation of a luminescent glow signal. The substrate (Z-DEVD-Aminoluciferin, Sodium Salt a Firefly substitute) DEVD peptide is cleaved, and the liberated aminoluciferin reacts with luciferase to generate measurable light. Caspase-3 and -7 recognises the DEVD tetrapeptide sequence.
Hi Nur,
In my experiments I also used Caspase Glo 3/7 from Promega after treating cells with proteasome inhibitor. In my case, I also noticed unhappy cells 24 hrs after treatment with inhibitors. Then I detected the Caspase activity after a series of increasing duration i.e. 6, 12, 18 and 24 hrs after treatment with inhibitors and found reasonable results.
Please go through my paper in the following link to get detail info..
https://www.ncbi.nlm.nih.gov/m/pubmed/28980748/
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