[Cytometry] PI intensity moves during analysis

Philip Hexley philip.hexley at hotmail.com
Tue May 10 09:51:53 EDT 2011


Hi Lizzie, 
 
Someone will probably enlighten us on the list as to the exact reason why but from what I understand it is from the change in pressure. I have always seen it and for that reason always accounted for it: running on analysers with 3/4 PSI it generally takes a few seconds so for the PI signal to stabilise, on the Aria I assume it is higher pressure so maybe this is why it is taking a minute to stabilise...
 
Cheers,

Phil
 
Phil Hexley
Flow Cytometry Facility
Shriners Hospital for Children - Cincinnati
 
> Date: Tue, 10 May 2011 14:35:10 +0100
> From: Elisabeth.Freyer at hgu.mrc.ac.uk
> 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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