Apoptosis assay: Cytochrome C release or Caspase-3 activity? - (Jan/04/2007 )

Hi-

Which is more definative of apoptosis: mitochondrial cytochrome C release or Caspase-3 assay in cell lysates?

So far I have PARP cleavage (WB) and BAK upregulation (WB) and 4-5 fold increase in apoptosis as measured by degraded DNA released into cytoplasm detected by ELISA (Roche cell death assay) oh and a lack of increase in cell number.

I plan to use Z-VAD next week to show inhibition of apoptosis.

I want to do one more assay to be absolutely sure... but I have very little $$$ and time so I want to choose the better assay

any thoughts?

-nancyd-

I think caspase-3 assay would b better.

-scolix-

QUOTE (nancyd @ Jan 4 2007, 08:59 PM)

apoptosis should not be demonstrated by a single method; so, use both in addition to the other tests you have mentioned; caspase 3 is commonly used but not all apoptotic ways use caspase 3

-The Bearer-

Agree with kosmodrom. Different apoptotic pathways vary too much in their requirement for different factors, you can't use just one assay. Some pathways don't result in significant DNA fragmentation, some don't give PARP cleavage etc. Plus, there can be cell-specific effects e.g. MCF7 cells don't express caspase-3, so no point looking for caspase-3 activation.
For my money: Best definitive assay, particularly if you have access to FACs machine, is Annexin-V staining (can use either FITC-conjugated Annexin-V or anti-phosphatidylserine-FITC antibody). Limitation is that it can only define early apoptotic cells, but I don't know of any kind of apoptosis where you don't get early exposure of PS on the outer plasma membrane.

-Hooly-