Calcium flux

Mario Roederer ROEDERER at Beadle.Stanford.EDU
Mon Jan 31 15:20:05 EST 1994

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.
(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 

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 at Darwin.Stanford.Edu

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