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Cleaved Caspase-3 for Flow

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#1 eigthball



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Posted 27 March 2013 - 08:51 AM

Dear y'all,

I'm new to this forum which I found (like most of you I guess) out frustration at the bench. I'm trying to stain cells for apoptosis (U2OS cells), which I treat with X. I'm trying a cleaved- Cp3 antibody that works great in IF (the Cell Signaling Technologies 9117 mono). It seems to work fine when I treat cells with staurosporine as a control, get 70%-90% stained cells. But when I treat with X, nothing. Except that X works great, I've seen it many times in IF, lots of dead cells. I only want to do flow for quantification. One big difference I've noticed between staurosporine and X is that staurospo-treated cells die but remain mostly intact. X-treated cells activate Cp3 and then break down very quickly in many small fragments (I have even made movies of them, it happens under 30 minutes. They don't even detach and float, they just "explode"). Is this a well-known issue when looking at apoptosis by flow? Is there any arcane flow way to get around this?

Any help will be much appreciated,


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