[Cytometry] best way to adjust PMT voltages?

helene barcelo barce012 at umn.edu
Mon Feb 23 23:37:53 EST 2009

Hi All,

I am running an assay measuring nucleotide excision repair on an LSR II (see
paper here:http://mutage.oxfordjournals.org/cgi/content/full/22/2/147). We
irradiate immortalized lymphocytes with UV and stain them with acridine
orange to measure the ratio of ssDNA v dsDNA.
In the assay, we include CEN in each tube as an internal control to check
for acridine orange staining in between samples and between days. We have
started to notice variations in staining intensity of our CEN (old and new
batch of CEN from biosure). This is not due to difference in our acridine
orange solution and we are suspecting that the slight change in CEN staining
is due to the slight variations of the instruments. Since we do quantitative
flow cytometry, we need to minimize the effect of instrument variations on
our experiment. So far I have been using constant PMT voltages for my red
(ssDNA) and green channels (dsDNA) So I am trying to find the best option to
take the instrument/laser variations out of the equation.
I think I have two options. I have been thinking about using CS&T and
application settings in the DIVA software to adjust the PMT voltages. And I
am wondering if any of you have had experience in standardizing assays using
that. My second option would be to use the CEN to adjust the PMT voltages by
keeping them in the same channel for each experiment. I will be testing
these options next week but I figured that you might have insights/advice or
way I can deal with this problem. Am I missing something?

I am looking forward to your answers. Thanks

Helene Barcelo
Lab Medicine and Pathology 1-350 Moos Tower
515 Delaware Street SE
Minneapolis, MN 55455
Office phone: 612-626-3341
Email: barce012 at umn.edu

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