Questions about apoptosis assays
peter_frankde at yahoo.de
Thu Jul 1 15:20:56 EST 2004
I would like to perform an apoptosis assay on two differently treated
human cell cultures. The cells are adherent and form monolayers.
By doing some research on the web I think a caspase-3 activity assay
might be appropriate in my case. Has anyone used such an assay before
and made good or bad experiences with it?
One problem is that the differently treated cell cultures also show
considerable differences in proliferation. So, even when I seed out
the same numbers of cells I won't end up with the same numbers after
the cells have grown up and the assay will be performed.
How do you normalize a caspase-3 (or any other caspase) activity assay
(or any apoptosis assay working with cell populations)? Do you count
your cells (how do you do that in a normal cell culture dish with no
microscopic grid?) and then normalize for cell number or do you make
cell lysates and then normalize for protein content (will the cell
lysis buffer components negatively affect the caspase-3 activity or
will the whole lysis procedure produce wrong results?)?
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