use of hypotonic PI solution to ID apoptotic cells
kweber@vs4.im.med.umich.edu
Thu, 1 Jun 1995 10:15:41 -0400
We are trying to use a hypotonic PI solution to identify apoptotic cells, and
we would like some input from anyone who has tried something like this.
The method is to incubate cells in this solution from 4 hours to 4 days at 4 C.
There is no ethanol fixation step. The solution contains 0.1% sodium citrate,
0.001% Triton X, 500 ug PI, and 500 ug RNAase. We ran the cells twice, about 14
hours apart; they were kept at 4 C except for 4 hours while they were being run.
We observed that the distance between sub G0 and G0 peak increased and the %
sub G0 increased. Also, on the first day, there were 2 distinct, but close,
peaks (sub G0 + G0 or G0 + synch S), and on the second day the peaks were
indistinct. This was inconsistant; not every sample showed a change. The
changes occured whether the cells were prepped for one day or 5 days. We would
appreciate any opinions on this protocol and any ideas on why these
observations are occuring. FYI, these experiments were run with Human B cell
lymphoma lines.
TIA
Melissa Tuck
This is Kris speaking now...
A few more details..
The protocol was published by Nicoletti et al, J Immunol Meth 139(1991) 271-279.
One additional note of interest, the article says that this protocol generates
a nuclei prep and it is clear from light scatter that we did not have a nuclei
prep. Maybe this is the source of the problem. Thanks for any and all help.
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