[Cytometry] Propidium staining

Anna Petrunkina ap542 at cam.ac.uk
Thu Feb 11 05:32:01 EST 2010


Dear Snelal,

what is the stock concentration of PI (in microg/ml or mmol)?
2 microl could be too much or too little, depending on your stock.

If you are not washing (or washing is not effective enough) and excess of 
PI is present in the solution, PI signal will change if and the cells are 
continuing to dye or undergo degenerative changes, so more cells will 
become positive. Besides, PI needs some time to get into cell, so 3 min 
could be just too short (we mostly leave 5 min).
I would suggest to check PI concentration first, and then do a kinetics 
test to see when a plateau is reached. An interesting paper to look at is 
Cytometry A. 2004 Jul;60(1):63-72, linking development of intermediate 
populations for SYBR14/PI staining to treatment and concentrations.

Hope that helps,
Best wishes,
Anna


Dr. Anna Petrunkina
Head of Flow Cytometry
Room 6.25a
Cambridge Institute for Medical Research
Wellcome Trust Building
Addenbrooke's Hospital
Hills Road
Cambridge
CB2 0XY


--On 10 February 2010 09:47 +0530 snehal mhatre <snehalrm at gmail.com> wrote:

> 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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