[Cytometry] PI intensity moves during analysis

Rossi Ralph ralph.rossi at petermac.org
Wed May 11 02:52:57 EDT 2011


 Hello All,
              I have seen this as well, to greater and lesser degrees
and SUSPECT it's some sort of equilibrium process
		  between the PI solution the cells are in and the
tubing/flow cell ?

ralph


Regards
Ralph Rossi
Flow Facility Manager 
Peter MacCallum Cancer Institute
Melbourne , Australia
Ph 96563747 , 96561955
Email : ralph.rossi at petermac.org

-----Original Message-----
From: cytometry-bounces at lists.purdue.edu
[mailto:cytometry-bounces at lists.purdue.edu] On Behalf Of Nebe-Von-Caron,
G
Sent: Wednesday, 11 May 2011 4:42 PM
To: Elisabeth Freyer; Cytometry at lists.purdue.edu
Subject: Re: [Cytometry] PI intensity moves during analysis

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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