High throughput 96-well format assay for apoptosis?

White, Damian D.White at genesis.co.nz
Sun Jun 17 21:59:06 EDT 2007


Fellow Flowers,

I'm in need of a high-throughput 96-well format assay for the DNA/cell
cycle/apoptosis analysis of adherent post-transfection populations.  New
to all things flow, I was in hopes that someone could point me in the
general direction of a kit I could buy in, or lend me some ideas as to
reagents and strategies for developing something in-house.

We're wanting to keep handling to a minimum, so I'm trying to keep away
from any manual lifting/mixing of cells.  Initially, I'm thinking of
washing the transfection population, then incubating cells with
Accutase/Accumax to lift/dissociate the population, with incubation time
to be optimised per cell type.	Then I'll add a secondary mix containing
a permeable DNA stain, incubate as needed, and acquire data.

Is this too simplific?  

I'm hoping that Accutase/Accumax treatment will be enough to generate
non-adherent single cell suspensions . . . a little investigatory
experimentation has shown that this looks to be the case with some cell
lines, maybe not so with others.  Could I incorporate some brand of
'counting beads' into my secondary mix, so that when I acquire data I
can get a measure of cell number?  Or would these been too 'sticky' for
a 96-well automated format that's going to take 2-3 hours to read?  And
if I were to add PI to that mix as well -- assuming I'll initially spin
the plate down so as to pellet any non-adherent/dead cells in the
population -- could I gate on these so as to get a relative idea of
viability in comparing PI exclusivity . . ?

Any help would be appreciated from a flow noobie.

:)




  Damian White
  Flow Cytometry Department
 
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  Genesis Research & Development Corporation Limited
  PO Box 50 Shortland St, Auckland 1140, New Zealand
 
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  Tel: +6493735600 DD:+6493743724 Mb: +6421377870

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