I use this double staining as a possible alternative to the expensive annexinV/PI staining after having set that usually the YOPRO-1 staining cells are slightly stained with A+/PI.
The question is that very often with toxic molecules, The YOPRO-1 staining is absent whereas I have a PI intermediate population of cells which I assume are permeable... What is teh matter of that.
Please find a flow histogram linked for analysis and help me please!
Dear Patrice
As far as I know Yo-Pro-1 is used as a nuclear label taken up by a channel/pore-forming P2X7 receptor. P2X7 receptor activity is dependent to a prolonged exposure to ATP which induces a second permeability state, which allows the uptake of larger cations such Yo-Pro-1 (1). A range of downstream events follow P2X7 activation, including phosphatidylserine (PS) exposure and cell lysis.
So I would have thought that the Yo-Pro-1 uptake is a step earlier to PS exposure which is really not the case in your experiments. My question would be which kind of cells are you staining? hematopoietic cells? other phenotypes? As we recently found that flippase activities on the hematopoietic phenotype are reacting differently from pancreatic or hepatic cancer cells (submitted paper)...
(1) Lopez-Castejon G, Young MT, Meseguer J, Surprenant A, Mulero V. Characterization of ATP-gated P2X7 receptors in fish provides new insights into the mechanism of release of the leaderless cytokine interleukin-1β. Mol Immunol 44: 1286–1299, 2006.
Your answer is very documented indeed but I was speaking about simple membrane permeability. The problem is not the correlation or not with a+/PI (even if useful) but the fact that I have no YOPRO_1 staining when PI is intermediate and tell me that the cell are permeable to bigger molecules. Can I say that the DNA is not accessible to YO_PRO_1. Could the DNA be already condensed ?
Dear Patrice, I was wondering if you had compensated the YOPro-1out of the PI channel? I think this is your problem, doing this should then give you YoPro-1 single positive data i.e. apoptotic cells, see the link to my website http://www.icms.qmul.ac.uk/flowcytometry/uses/apoptosis/diagrams/YOPRO.jpg
Regards
Gary
Do not worry, I effectively known the problem you are talking about. But my settings are good. But, the situation the one I described.
Patrice
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