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(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.
(3) Cells are then washed at room temp 2-3 times to wash out the azide,
and resuspended in medium at room temp. All washes and suspensions are
done in complete growth medium, or just RPMI with 4% serum.
(4) Cells are warmed to 37C for 5 min prior to baseline and stimulation
collections.
I have stained with: CD4, CD8, CD45RA, CD45RO, and L-Selectin. Various
controls have demonstrated that this staining protocol does not
significantly alter the peak calcium, the final calcium, nor the
kinetics--with the possible exception of CD8 causing the CD8 T cells to
flux somewhat better. We will be publishing this shortly in Cytometry.
Finally, I used Consort-VAX to analyze the huge files that arise from
these experiments. (Typically, we collect 8 parameter data at 2000
cells per second for up to 15 minutes). Consort-VAX works quite
efficiently and has shown no problems to date. By the way, I have
rewritten the kinetics analyzer for Consort-VAX to work with files that
have TIME as a parameter rather than storing kinetic information the way
that Consort-VAX does--if anyone is interested, please contact me.
Mario Roederer
Roederer@Darwin.Stanford.Edu
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