Hoechst 33342 DNA staining

Hi, could anyone provide advice for my colleague Michael who is having
trouble with Hoechst 33342 staining - thank you: Bridget


I've been trying to stain and sort live, unfixed peripheral blood
mononuclear cells (PBMC) using Hoechst 33342 DNA staining and have
encountered a major problem. After a 60-90 minute incubation in a 37
degree/5%CO2  incubator, I have found the cells immediately begin going
down in fluorescence intensity while running on the cytometer
(presumably because the cells begin to pump out the dye). The result is
a DNA histogram that is constantly moving with a very bad coefficient of
variance (CV) because of the shifting. In sorting (by constantly
adjusting the PMT) and then fixing and staining with PI, I have been
able to get pure G0/G1 populations but S and G2/M populations are only
about 50-65% pure. I have found chilling the cells actually increases
the rate of dropping in fluorescence and have since then have been
taking the cells out of the incubator just before running the sample on
the cytometer.


Has anyone encountered this same problem with Hoechst 33342 and found
any "tricks" to reduce this rate of drop in fluorescence in live cells?


Thanks,

Michael Trifiro

University of California Davis

Internal Medicine: Dept. of Infectious and Immunologic Diseases