[Cytometry] PI intensity moves during analysis

W.E.Corver at lumc.nl W.E.Corver at lumc.nl
Tue May 10 13:36:26 EDT 2011


Dear all,

I might be wrong but I agree with Phil that it is likely caused by less stable hydrodynamics (small pressure fluctuations) at the start of the analysis. Using a metaphor: it is just like driving your car through a bump in the road. You can still feel the car shaking a few seconds after passing the bump. The same might happen during the start of the acquisition. There is a sudden pressure increase causing a wave which is dampened. The phenomenon is somewhat more notorious on the ARIA I with the relatively old plenums. Using the same samples you hardly see it on an ARIA SORP.
Since the interaction between the sheath and the staining buffer is a continuous process starting at the entrance of the cells or nuclei in the flow chamber (ARIA) I would like to suggest that this will have an effect on the CV but is not responsible for the signal increase during the start of the measurement. You can see the affect Zbigniew is referring to when you change the sheath for water running Vindelov samples. Scatter characteristics are distorted and CVs are increased.
Concerning methanol versus pepsin (Hedley) and Vindelov method: the latter two methods might introduce free DNA in the sample which will have an effect on the viscosity. Secondly, Hedley and Vindelov make use of bare nuclei rather than “intact” cells, which might be more stable in flow due to their relative large size and mass.

Kind regards,

Willem Corver

p.s. Elisabeth you better use a 610/20 BP for collecting PI fluorescence.
________________________________________
Van: cytometry-bounces at lists.purdue.edu [cytometry-bounces at lists.purdue.edu] namens Darzynkiewicz, Zbigniew [Z_DARZYNKIEWICZ at NYMC.EDU]
Verzonden: dinsdag 10 mei 2011 17:25
Aan: 'Philip Hexley'; elisabeth.freyer at hgu.mrc.ac.uk; cytometry at lists.purdue.edu
Onderwerp: Re: [Cytometry] PI intensity moves during analysis

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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> Cytometry at lists.purdue.edu
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