ps-pi staining

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rahi
rahi's picture
ps-pi staining

I do PS-PI staining of my cells to chk whether the stressed cells are undergoing apoptosis or necrosis but the control cells (without any stress) show pi positive. what could be the reason?

jonatmudd
jonatmudd's picture
are you sure that your non

are you sure that your non-stressed cells aren't undergoing either nec. or apop.? maybe the stain is hurting your cells.
i'm not sure what cells you are staining, but if you aren't careful with matching osmolarity before and after staining, you may be severely stressing cells. seems to be a pretty common and easy mistake to make.

maybe this is stating the obvious, but sometimes it is easy to overlook the obvious.

ltrbrown
ltrbrown's picture
How long do you wait to

How long do you wait to analyze after the addition of PI? I stain for PS according the protocol I have for AnnxV-PE (BD). Then I add PI (final 5ug/ml) or 7-AAD (final 1ug/ml) to the cell suspension (in PBS) just before analysis... literally 2-3 min. This is where I deviate from the manufacturer's protocol, which reccomends a 30 min incubation with PI. I feel that this is far too long to assess viabilty... necrotic cells should be almost immediately permeable. Due to the toxic nature of these dyes I think they will eventually "permeate" on their own which could lead to non-specific death, especially in fragile cell types.

hinaarehman
hinaarehman's picture
ltrbrown wrote:How long do

ltrbrown wrote:

How long do you wait to analyze after the addition of PI? I stain for PS according the protocol I have for AnnxV-PE (BD). Then I add PI (final 5ug/ml) or 7-AAD (final 1ug/ml) to the cell suspension (in PBS) just before analysis... literally 2-3 min. This is where I deviate from the manufacturer's protocol, which reccomends a 30 min incubation with PI. I feel that this is far too long to assess viabilty... necrotic cells should be almost immediately permeable. Due to the toxic nature of these dyes I think they will eventually "permeate" on their own which could lead to non-specific death, especially in fragile cell types.

I analyse the cells just after addition of PI at 5ug/ml concentration