I don't classify myself as a cell-cycle guru, but did some of
that in a previous job.
Some suggestions:
-I would always include some reference cells as an internal
standard, these are ideally pre-mixed and go through the same
prep and staining procedures as the cells of interest.
-Adjust the cell concentration after washing but before PI
staining.
I think if you checked the samples that are shifted you will
probably find a marked difference in the cell concentration
from the others.
In the context of screening lots of tumour samples, it took
a long time to convince a certain person that these variations
meant we could not differentiate between hypo- and hyper-
diploid.
Hope this helps a little.
Joseph.
At 22:55 4/05/2000, Claudio Vallan wrote:
>A probably stupid question for the cell-cycle gurus:
The times I felt most stupid were when I didn't ask...
>When doing cell cycle analysis (PI stained cells) here and then I have a
>sample which for no apparent reason shifts in staining intensity when
>compared to the others. How do you adjust for this if you do not use such a
>program as Modfit to calculate the percentages of the different stages? Do
>you correct the PMT voltage during aquisition in order to get the G1 peaks
>at the same positon or do you change the position of the markers at
>analysis (Or do you throw away the data of those samples)? If you change
>the markers, do you do do this just by eye or do you use a certain
>algorithm to do this? Is somebody including references as RBC nuclei?
--
Joseph Webster, Flow Cytometry Facility
Centenary Institute, Sydney AUSTRALIA.
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