[Cytometry] PI intensity moves during analysis
g.nebe-von-caron at alere.com
Wed May 11 02:42:03 EDT 2011
If you reload the same sample a second time do you get the same change
Research Scientist and Biomedical Engineer
From: cytometry-bounces at lists.purdue.edu
[mailto:cytometry-bounces at lists.purdue.edu] On Behalf Of Elisabeth
Sent: 10 May 2011 14:35
To: Cytometry at lists.purdue.edu
Subject: [Cytometry] PI intensity moves during analysis
I am observing some strange shifts of the PI intensity when i do cell
cycle analysis. I'm not talking about the shifts between samples, my
whole histogram of the same sample actually moves up in intensity
within the first minute of running it on the machine. After a minute
it stays about the same.
I use an Aria2, measure PI in 488-685/35BP
It ONLY happens when I use protocols that require treating the cells
with Pepsin or Trypsin (e.g. Vindelov) prior to PI staining. no
movement w/ EtOH fixation and staining in PI/RNAse solution only.
The samples are sitting on ice until they get loaded onto the machine.
I thought it might be temperature related, but also with a cooled
sample chamber it still moves.
To record I now usually wait for a minute for the PI intenisty to
stabilize and record then. It gets a bit difficult when I have samples
with very small cell numbers :(
I'd greatly appreciate if somebody could enlighten me where that comes
from and if there is anything I can do to stop this.
Flow Cytometry Facility
MRC Human Genetics Unit
Western General Hospital
Tel: 0131 332 2471
email: Elisabeth.Freyer at hgu.mrc.ac.uk
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