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I am interested in your comment that if PBMC are put on ice, they will
not "Ca++ flux", even apparently after warming to 37 deg for 5 min.
Are you aware of any published data on this point, or on any other
signal/messenger hysteresis after ice?
Is the azide just there because your antibodies are in it, or is it
there for a reason as far as the PBMC are concerned?
In message Mon, 31 Jan 1994 13:20:05 -0700 (PDT),
Mario Roederer <ROEDERER@Beadle.Stanford.EDU> writes:
> We have been doing calcium flux on unseparated, stained PBMC. The
> protocol we use is pretty standard, with the following important points:
>
> (1) Cells are loaded at 37C with Indo for 45-60 min
> (2) Cells are centrifuged and then spun at room temperature in the
> presence of 0.02 to 0.1% sodium azide with antibodies. We use only
> direct conjugates to avoid crosslinking. It is EXTREMELY important not
> to let the cells get too cold (never on ice); they will not flux.
Thanks,
/*- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
Eric Martz, Professor of Immunology emartz@microbio.umass.edu
Dept Microbiology Voice: 413-545-2325 FAX: 413-545-1578
Morrill IVN 203, Box 35720, Univ Massachusetts, Amherst MA 01003-5720
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