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I'm trying to establish the Calcium Flux protocol for BMDCs dendritic cells in my laboratory. I've gotten a few results, but I'm unsure if they are showing calcium fluxes or otherwise.
An example of my result is this:
(using BDFACSDiva)
(using FlowJo)
I'm expecting to see this:
But, no matter how quick I try to be, the first image is all that I get. So, my question is is the first image considered to be a flux. Or is it because of the high cell count as I re-installed back the tube. I'm using 2 tubes for 1 run; one to get the baseline, and the other with the agonist added. This is to ensure that the cells are at 37°C when I add the agonist. Number of cells per tube is 1 x 10^6 cells.
I'm using Indo-1 dye, and doing my analysis with BD LSRII. My filters are blue 530/30 and violet 405/20. The voltages for indo-1 blue is about 600++ while that for indo-1 violet is 500++. Should I raise them for a better result? I'm asking this as I'm using DMSO to dissolve the dye, instead of anhydrous DMSO as recommended by the manufacturer. But, I've compared the results of 1 week old dye and freshly prepared dye and they're similar, so I think the dye should be stable in normal DMSO.
Any help or advise is appreciated.
