Cell Death. optical spillovers.alternative assay - Need an assay for apoptosis (Aug/09/2005 )
well i am now into 3 months monitoring my cells for apoptosis using a novel small molecule inhibitor. the problem is that this inhibitor has a inherent red color. when used in FACS for PI staining the signal from the drug overlaps too much over the PI and the signal from the PI is hardly compensated.
When i did an annexing FITC staining...the emission of drug was so much that it spilled over and into the FITC channel.
so now i did a caspases asssay(BD Apoalert), there was also a small background from the drug, which increases with incubation period.
All the above techniques could not generate a valuable(publishable) data...but i know that theres apoptosis taking place.
is there ne other alternative assay on the list?? MTT assay looks good but its also has trouble with a red emmission.
could u suggest anyother technique to solve the problem.
Assuming your apoptosis is caspase-dependent, you could try a caspase 3/7 assay. They are made in both fluorometric and colorimetric varieties. Alternatively you could do westerns for cleaved caspase 3, or for cytochrome c release from the mitochondria into the cytosol. I have never had luck with the cleaved caspase 3 antibody, though.
You can also check for nuclease activity by looking for characteristic DNA laddering in your apoptotic cells.
There are other supposed markers of apoptosis like histone modification, but I don't really buy into it. Regardless there are many alternative assays you can do that your inhibitor will not interfere with.