Re: Annexin V and PI staining for apoptosis

Dear Li,
I forgot to mention one more possible reason for AnnV-PI+. If there is a deficit of
bivalent cations in cell suspension media while running on flow, cells may loose
previously bound annexin while being still stained with PI. For example, if you do not
keep cells all the time after staining in annexin binding buffer, but wash them and
resuspend in some Ca++/Mg++ free solution, like PBS.
The flow cytograms with AnnV- events that I have seen did not suggest the above reason by
the look of event populations, however I think, in theory, it is a possibility.
Regards.
Vladislav Rozenkov, M.D., Ph.D.
Dear Li,
Cell population No.4 should not exist by definition, since PS becomes available to
annexin V before cell membrane becomes permeable to nuclear stains (PI, 7AAD).
Therefore, as others have noted, the events in population 4 area are most likely not
cells, but nuclei or particles. It may help if you try to back-gate these events onto
scattergram (and any other histograms that you have) and see whether you can pre-gate out
these events by their size or other characteristics.
Make sure you are not using baseline offset!
We also sometimes see AnnV-7AAD+ events (unfortunately), but ideally they should be
either absent or negligible. The latter means that a few events seen in that quadrant may
be ignored if their percentage is low among the cells in other quadrants. 
Otherwise, you need to exclude these odd events, as you do not know what particles they
are - nuclei or some pieces, i.e. several events from one destroyed cell. This is to
answer your second question.
I have noticed that the 7AAD or PI "brightness" of these AnnV- events is quite high,
comparable to double positive cells, which supports the idea that they are nuclei
stripped of cytoplasm and cell membrane.
Regards.
Vladislav Rozenkov, M.D., Ph.D.
-----Original Message-----
From: chen li <chen_li3@yahoo.com>
To: cyto-inbox
Sent: Wed, 25 Jan 2006 16:18:30 -0800 (PST)
Subject: Annexin V and PI staining for apoptosis


Dear Flowers,

    When Annexin V and PI are used for staining cells
and subjected to FLOW there are 4 populations
(sometimes 3), which are 1) Annexin V and PI both
negative (alive cells), 2) Annexin V positive only
(cells at early stage of apoptosis), 3)Annexin V and
PI both positive (late apoptotic cells) and 4) PI
positive only (???). I couldn't find the explanation
for PI positive only. 

    My questions are 1) what happens to PI positive
only population? 2) Is this population included in the
percentage of apoptosis cells?

Thank you very much,

Li  

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