[Cytometry] Propidium staining

snehal mhatre snehalrm at gmail.com
Tue Feb 23 09:48:42 EST 2010


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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