Hi!
I trying to block bcl-2 in bcl-2-overexpressing cell culture model. In my conditions ABT take effect at 2 mkM - cells turns apoptosis and dead. But ABT-199 active at nanomoles. What concentration you use and which way to confirm bcl-2 inhibition?
Thanks!
Some cell lines are resistant to ABT 199 so I believe the concentration needed varies from cell line to cell line, specially since you are working with Bcl2 overexpression. I found an article that shows concentrations ranging from 500nM to 2000nM (90% inhibition). As for the best way to confirm I believe you should go for western blot considering that Bcl-2 is degraded after ubiquitination.
http://www.bloodjournal.org/content/122/21/3978.full.pdf
Dear Serguei,
You should determine IC50 in your model using MTT or annexinV-PI staining. In some cells IC50 for ABT-199 can be in LOW nanomolar conc.
I don't agree with Andre, you will not see any decrease in Bcl-2 levels after ABT-199, it doesn't work that way. What ABT-199 does it competes with proapoptotic BIM in binding to Bcl-2. After ABT-199 is added, BIM can be released and forms a complex which destroys mitochondrial outer membrane.
The readouts of Bcl-2 inhibition after ABT-199 usage can be:
cyt.C release from mitochondia to cytoplasm,
PARP cleavage
Caspase3/7 cleavage.
Please refer to enclosed paper
Best,
Kamil
Hi Serguei,
I agree with Kamil answer. You need to determine IC50 in your cell culture using some assay to measure citotoxicity. In some cells IC50 for ABT-199 concentration can vary between micro and nanomolar. You must to do the citotoxic assay to know what concentration is optimal for your cells.
Good luck,
Joan
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