[Cytometry] PI intensity moves during analysis

Darzynkiewicz, Zbigniew Z_DARZYNKIEWICZ at NYMC.EDU
Tue May 10 11:25:27 EDT 2011


Hello Lizzie and Phil,
Staining of DNA with dyes such as PI, 7AAD or PI is done under conditions of equilibrium between the dye and DNA. It is a subject of chemical mass action law and also affected by ionic interactions of the salts present in the staining solutions. There is a change in dye (and salts) concentration due to dye (and ions) diffusion from sample flow to sheath flow and it takes time to establish the stable equilibrium. Depending on geometry of the channel and length of the flow stream until it meets the laser beam the equilibration time may be of different duration. We described there interactions in the chapter which is free to access: 
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2967208/?tool=pubmed

and also in the Current Protocols in Cytometry 

http://www.ncbi.nlm.nih.gov/pubmed/21455968



Zbigniew

Zbigniew Darzynkiewicz, MD, PhD.
Professor of Pathology and Medicine
New York Medical College
Valhalla, NY.

Department of Pathology
15 Dana Road
Valhalla, NY. 10595
Tell. 914-594-3794, 3780
http://www.darzynkiewicz.com/zbigniew/



-----Original Message-----
From: cytometry-bounces at lists.purdue.edu [mailto:cytometry-bounces at lists.purdue.edu] On Behalf Of Philip Hexley
Sent: Tuesday, May 10, 2011 9:52 AM
To: elisabeth.freyer at hgu.mrc.ac.uk; cytometry at lists.purdue.edu
Subject: Re: [Cytometry] PI intensity moves during analysis


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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