We have found that a majority of CD4+CD8+ cells, especially after
either freeze/thaw or stimulation, are actually dead cells.
Unfortunately, dead cells are often "sticky" and bind antibodies
non-specifically--thus, becoming positive for all antibodies
("CD3+CD4+CD8+"). When a viability marker (such as PI) is used, most
of these can be excluded to reveal live cells. Among live cells, the
actual incidence of DP cells in most healthy adults is quite low,
less than 1% of T cells.
For use with intracellular cytokine staining, you would have to
employ EMA (ethidium monoazide), a viability marker which survives
the fixation/permeabilization steps. EMA also helps excluded tons of
apparent cytokine positive cells that are actually also dead!
Look through EMail archives for previous discussions on this topic
for references etc.
mr
At 6:05 PM -0400 8/25/00, Liza McGuire wrote:
>Hi Flowers,
>
>This time the question refers to CD4+CD8+ cells?
>Has anyone observed a significant double positive population on
>Neupogen stimulated PBSCs? The cells are CD3+/CD4+/CD8+ of which 10%
>of the total CD3+ cells are CD4+CD8+. We gave a quick look at the
>scatter of the cells and they are the same size as all T cells
>(smack in the middle of the lymph gate). We looked at the cells
>morphologically by staining them and could not see any immature
>and/or abnormal cells (normal diff). Any ideas? I appreciate any
>thoughts on the matter.
>Thank you,
>Liza McGuire
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