[Cytometry] Propidium staining

Gerstein, Rachel Rachel.Gerstein at umassmed.edu
Tue Feb 23 12:54:41 EST 2010


hi,

you might want to make aliquots and store your stock solution of PI in the freezer and then dilute what you need for each day and use it fresh at 1 ug/ml [final concentration with cells].  in your lysed PBL prep,  when you run with and without PI, the neuts but not the lymphs are PI+ ? how are you identifying neuts and lymphs ?
=======================================================
Rachel M. Gerstein, Ph.D.
Associate Professor
Department of Molecular Genetics and Microbiology
Graduate Program in Immunology/Virology
University of Massachusetts Medical School
55 Lake Avenue North
Worcester, MA 01655-0002


________________________________________
From: cytometry-bounces at lists.purdue.edu [cytometry-bounces at lists.purdue.edu] On Behalf Of snehal mhatre [snehalrm at gmail.com]
Sent: Tuesday, February 23, 2010 9:48 AM
To: Barry Moran
Cc: cytometry at lists.purdue.edu
Subject: Re: [Cytometry] Propidium staining

Thank you so much for your suggestions.

Actually PI which i was using was prepared few months back and i think due
to evaporation, the solution had become concentrated. so i prepared fresh PI
solution and now it is working well for lymphocytes. but my neutrophiles are
showing 100% non-viability. I am using fresh human whole blood, lysing with
ammonium chloride lysing solution without fixative and then cells are
stained using 2uL of 0.5mg/ml PI solution.
What could be the reason for it?

Thank you.

On Thu, Feb 11, 2010 at 3:15 PM, Barry Moran <barry.moran at tcd.ie> wrote:

>  Hi Snehal
>
>
>
> I find PI gets into non-viable cells almost instantaneously, so you dont
> have to wait the few minutes or spin down again etc. I use 1ug/ml final
> volume of PI (not clear what conc. “2ul” is)- perhaps you’re using it far
> too concentrated and background is changing. Also, best to look at the
> fluorescence in log scale (unlike for example when looking at cell cycle
> using PI, on lin scale). FL3 is probably right, though I’m not sure what
> cytometer you’re using (channel filters vary between machines).
>
>
>
> It should be fairly straightforward then- all non-viable cells should
> fluoresce high (maybe 100 fold brighter) at ~620nm (probably in FL3 on your
> machine); viable cells should be basically same as background.
>
>
>
> All the best- let me know if you have any further questions.
>
>
>
> Barry.
>
>
>
>
>
> Barry Moran
>
> Research Officer and Manager
>
> Flow Cytometry Facility
>
> School of Biochemistry and Immunology
>
> Lab 1.8, Biochemistry Building
>
> Trinity College Dublin
>
> Ireland
>
>
>
> Lab:    +353 1 896 2761
>
> Mob: +353 87 921 0811
>
>
>
> barry.moran at tcd.ie
>
>
>
> http://www.tcd.ie/biochemistry/flow
>
>
>
>
>
>
>
> -----Original Message-----
> From: cytometry-bounces at lists.purdue.edu [mailto:
> cytometry-bounces at lists.purdue.edu] On Behalf Of snehal mhatre
> Sent: 10 February 2010 04:17
> To: cytometry at lists.purdue.edu; cytometry at flowcyt.cyto.purdue.edu
> Subject: [Cytometry] Propidium staining
>
>
>
> I have to stain my cell line (K562) with PI (Propidium Iodide) to check the
>
> viability. I am adding 2uL of PI and incubating it at Room temperature for
>
> 3mins and the aquiring on flow cytometer without washing. The peak is
>
> shifting. So, i tried it with freshly isolated human PBMC but I am getting
>
> the same results.
>
> I even tried giving it a wash before aquiring, but it didnt made any
> change.
>
>
>
>
>
> I m aquiring PI in FL3.
>
>
>
> Can anyone help me out with this? I wanted to know is my protocol is right
>
> or can anyone suggest me any changes.
>
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