PI and human lymphocyte cultures - (Oct/18/2005 )

Hi, my name is Marcela. I am working with human isolated lymphocytes in order to observe their
death processe induced by certain drugs (nitroimidazoles). I expose the
cells during 48 hours at 37ºC in humidid incubator with 5% CO2 using
RPMI 1640 medium, Fetal Calf Serum (5%),1x106 cells/ml and 10, 50 and
100 ug/ml nitroimidazoles final concentrations. Moreover, others cells
are exposed to dexametasone as positive control. After that I try to
detect hypodiploid cells by PI assays using flow cytometry. Twice I
obtained positive results for both nitroimidazole drugs and
dexametasone. But since then it was imposible for me to get positive
results again, whether the positive control or the nitroimidazoles.
I do not know which is the reason for those inconsistent results.
Does anybody know what is happening?
The cells keep alive and they do not show any change. The cytometric
results of the treated cells are the same as the untreated cells.
I would like to receive your opinions.
Thanks.
Marcela.

Hey Marcela,
The possible reasons for inconsistent results could be differences in cell numbers before treatments. or the PI reagent/ PI containing buffer is not fresh. try keeping the number of lymphocytes around 0.5 mil when u start the treatment. and prepare the reagent fresh

All the best