induce necrosis

Janet E. Lewis jelewis1 at facstaff.wisc.edu
Mon Mar 23 00:25:40 EST 1998


I found that microwaving the cells for a few seconds (10sec on high for
cells in 10ml medium in a 10-cm diameter petri dish) did the trick.  They
were still intact for at least 2 hours (at 37C) afterward.  I didn't make an
extensive study of how the cells decayed over time.   I wanted annexin V-
and merocyanin 540-binding positive, definitely non-apoptotic controls to
compare with HL-60 cells induced to apoptosis by camptothecin. I was looking
for a way to disrupt the membrane integrity without making the cells
immediately fall to pieces while simultaneously making sure the cells
wouldn't have any chance to activate any apoptotic enzymes. 

 The "nuked" cells gave nice, bright annexin V staining or merocyanin 540
staining.  If stained with PI for DNA content, they gave histrograms the
same as those from normal, untreated cells, with distinct G0/G1, S, and G2/M
regions (perhaps a "snapshot" of how the cells looked at the time they were
microwaved).

>What about heat your cells at 56 C for 30 minutes, in a matters of hours
>you will see the dead cells and it seems that is not by apoptosis since
>there is not blebing.
>Cheers Rafael

>>Hello, I'm setting up an experiment to induce necrosis and apoptosis in
>>cell line. References I found on inducing necrosis involve maintaining
>>harvested cells, tightly capped, at 37oC and 5% CO2 incubator for 7-10
>>days; and using dexamethason to induce apoptosis. My question is if
>>anyone out there has a method for inducing necrosis that can be
>>accomplished within say a few hrs. Will appreciate any ideas. Thanks in
>>advance. Nancy Gin - United Hospital, St.Paul, MN
>

--Janet

University of Wisconsin-Madison
Comprehensive Cancer Center
Flow Cytometry Facility
Madison, WI
jelewis1 at facstaff.wisc.edu




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