Re: bulk removal of dead cells

Kevin G Waddick (waddi002@maroon.tc.umn.edu)
Thu, 19 Jun 1997 12:48:33 -0500 (CDT)

All you need to do is layer the cell suspension on top of Ficoll-Hypaque
and centrifuge it as you would to isolate MNC from BM or blood. I'm not
sure what you mean by "quantitative," however the method works well for us
when we want to remove dead cells from patient samples that were
cryopreserved years ago. To further separate the dead cells (necrotic and
late apoptotic) for FCM analysis of "alive" cells, use propidium iodide to
gate out the cells that stain. The cells that are beneath the PI-positive
cells that are furthest to the left (assuming that PI positivity is
measured on the vertical axis) are the early apoptotic cells; these can
also be gated out.

Kevin G. Waddick, Ph.D.
Staff Scientist
Hughes Institute
2685 Patton Road
Roseville, Minnesota 55113
(612) 628-0598
(612) 628-9891 Fax

On Thu, 19 Jun 1997, Ralph Bohmer wrote:

>
> Is there any perfect method to bulk isolate a small number of vital cells
> from a much larger population of dead and dying cells (apoptotic, necrotic,
> debris) ?
>
> It would have to be quantitative.
>
> There is lysis of dead cells by protease. Does that safely preserve all
> viable cells ? If yes, what are the best conditions ?
>
> I was told it can be done by density gradient, but I cannot figure out how
> that should work.
>
> Many thanks for your opinions.
>
> Ralph M. Bohmer
>

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