[Cytometry] Propidium staining

Barry Moran barry.moran at tcd.ie
Thu Feb 11 04:45:56 EST 2010


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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