PI in spermine buffer is used for cell cycle analysis by flowcytometry. I would like to know can this Solution be used for apoptosis analysis as well?
I have already used 5,10 and 15 ul of 50mg/ml of PI (in spermine) , stained cells for 1-10 min, but in some cases it seems most of cells are dead. Is there any possibility in interfering spermine and DNA of even live cells?
If it can be used what's the volume and concentration?
Hi,
do you want to do the apoptosis analysis via FACS or microscope? And what are the exact components of the spermine buffer?
As I mentioned, yes I do apoptosis by FACS. PI for cell cycle is diluted in spermine buffer instead of PBS. PI in PBS is used for apoptosis. I wanna know can Pi i spermine be used for apoptosis or no.
Hi Hamed.
Yes, the same staining and analysis procedure used for cell cycle analysis can be used to analyze cell death. You have to make sure you gate small size 'cells". This will give you the sub-G1 population, which you can count. The sub-G1 population is the fragmented cells that have died most probably through apoptosis.
Another way to do this is to co-stained with Annexin V. The annexin V-positive cells plus the double positive represent the apoptotic cells. The Pi-only cells represent cells that died by necrosis.
Good luck
Thanks dear daniel for answer. But I meant what's the differences between PI in PBS buffer and PI in spermine buffer.Can Pi in sperime buffer be used for apoptosis or not. My observation is that PI in spermine can not give accurate result for apoptosis as all cells are stained even in low dose of Pi and even in short time incubation.
Hi Hamed
I don't know the difference between those two conditions. I would assume it should be very similar. Staining all the cells should not be a problem as you want all of them to be stained and count the fragmented ones (the sub-G1). However, ti should be quick and easy to compare them if you have the reagents. That should definitely clear any doubt.
"Can Pi in sperime buffer be used for apoptosis or not."
The answer is yes. Although there might be other sources of information, this kit uses PI in Spermine buffer. You can see the analysis in page 1 figures B and C. https://www.bdbiosciences.com/documents/Cell-Cycle-DNA-DS-Kits-Templates.pdf
And you can see that the PI is in spermine buffer in page 2. Here is also the technical manual if you want to take a look at it. http://www.bdbiosciences.com/ds//is/tds/23-2923.pdf
I hope that helps
Best,
Daniel
Hi Hamed, you can use PI in spermine for death cell discrimination but using a lower PI concentration, you should decreased it 10 times, at least. Spermine can make plasma membrane more permeable to PI, therefore a lower concentration is used for cell death discrimination. I hope it helps, best regards, Alberto
Thanks Daniel for the great comment. Thanks Alberto for the valuable advice. Thank you so much.
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