Questions about apoptosis assays

Cornelius Fritsch c.fritsch at dkfz.de
Mon Jul 5 02:48:58 EST 2004


Get the cells detached and do FACS analysis, e. g. phosphatidyl serine
exposure, propidium iodide uptake, or FACS the nuclei directly for
hypodiploid DNA content (Nicoletti et al., I might dig up the ref.
somewhere)

Cheers,

Cornelius


"Peter Frank" <peter_frankde at yahoo.de> schrieb im Newsbeitrag
news:g8m8e0pr7rj0e883tq0m06td8pcr8mclcm at 4ax.com...
> Hello,
>
> 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?)?
>
> Peter





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