mouse peritoneal mast cells

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Dear Flowers,

        We are having difficulty with 'autofluorescence' of a cell
population in a flow study involving mouse peritoneal mast cells.  The
problem is not so much with the mast cells, but with whatever else comes up
in the peritoneal lavage.  Isotype background of the mast cells is clean,
and our positive signal is well into the third log.  Forward and side
scatter gating on the population of interest (heterogenous FSC / high side
scatter) includes a highly autofluorescent population (second log) which
may mask  intermediate / down regulated c-kit positive mast cells.
Isolation by density gradient resulted in low yields of mast cells (and the
risk of preferential exclusion of the population of interest);  attempts to
reduce autofluorescence by trypan blue (Cytometry v.30 no 3;  June 15 1997)
have not succeeded so far, and I doubt it could reduce it to the level
required.

        I would appreciate any ideas on how to effectively gate on the mast
cells, reduce autofluorescence of the 'intruders' or isolate one or the
other populations.

        Thanks, Claude

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