Annexin on PBMC - real or false positives?

Hi all,

I've been running various AnnexinV/PI experiments on fresh PBMC isolated
by leukapheresis, and keep getting very large percentages of the cells
positive for AnnexinV - anywhere from 25% to 65% depending on where I
set my region/quad/marker.

Two questions:

1. Has anyone seen false positives on PBMs (or in general) with
AnnexinV?

2. Where should the gate/quad/marker be set on Annexin stained samples?
At marker 1 or 2 on the enclosed plot? Ie should the gate be based on
marker 1 - anything positive as compared to cells unstained for AnnexinV
- meaning no protein background control? Or should the gate be set at
marker 2 with the assumption that there is some 'nonspecific' binding of
annexin to the negative cells (dimmer peak)? I have a hard time using
just autofluorescence as a negative control, but there doesn't seem to
be a good protein negative control for Annexin. I've enclosed a plot of
the staining for AnnexinV and for simplicity have just shown a
univariate plot for the Annexin staining. The histogram plot represents
cells gated on CD45+ / 7AAD-. The overlay represents unstained, ungated
cells. This sample is 30hr post draw stored at RT.

Details:
I'm running the tests using the Pharmingen/BD kit, using PE AnnexinV
with a 2.5mM Ca++ buffer, and am testing samples both 3 hr and 30 hr
post draw. I've run samples double stained for CD45 / AnnexinV / 7AAD or
CD14 / AnnexinV / 7AAD. All of my monos are Ann+/7AAD- and various
percentages of the CD45 cells are Ann+/7AAD-. Interestingly I've found
literature that distinctly says monos are surface negative for PS. The
cells are either untreated or platelets have been washed out. There is
no red cell lysis or gradient separation because of development SOP
limitations.

I've searched the literature and pucl archives and have found info on
Jurkat, CEM, thymocytes, Raji, tonsillar Bcells, peripheral blood
neutrophils, murine tcell hybridomas, HL-60, monocytes & macrophages,
but nothing on PBM's in general. I've spoken with BD tech support and
they gave me a poorly explained story about problems using Annexin with
PBMC because of the monocytes & red cells. My next step is to try a
caspase or tunnel assay, as one assay may not tell the whole story.

Apologies for the length of the post and thank you for any help,
Sasha Lazetic,
Anosys, Inc
 <<Ann staining for pucl.pdf>> 


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