[Cytometry] PI intensity moves during analysis

Nebe-Von-Caron, G 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
with time?


Gerhard Nebe-von-Caron
Research Scientist and Biomedical Engineer 

-----Original Message-----
From: cytometry-bounces at lists.purdue.edu
[mailto:cytometry-bounces at lists.purdue.edu] On Behalf Of Elisabeth
Freyer
Sent: 10 May 2011 14:35
To: Cytometry at lists.purdue.edu
Subject: [Cytometry] PI intensity moves during analysis

Hello everybody,

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.

Cheers
Lizzie
:)

-- 
Elisabeth Freyer
Flow Cytometry Facility
MRC Human Genetics Unit
Western General Hospital
Crewe Road
Edinburgh
EH4 2XU

Tel: 0131 332 2471
email: Elisabeth.Freyer at hgu.mrc.ac.uk

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