We read out Ca release from internal stores as response to stimulation of lymphocytes, based on increase of Fluo-4 fluorescence in FACS. The problem is that the basal level differs a lot between samples, should we subtract it from each sample or should we rather divide to get the "fold increase"?
Fluo4 data is often expressed as a ratio to the basal values (i.e., F1/F0). You can also calibrate the dye using calcium standards. Don Bers has published several excellent articles detailing how to do this - but it is not a trivial exercise and I do not know if it can be applied to FACS.
I would split each sample in two just before stimulating one of them, do the unstimulated half first, do the stimulated sample (presumably the stimulation takes some time) and subtract the basal-unstimulated state from the excited state. There are many reasons for varying basal levels. For example, it is very easy to stimulate the lymphocytes just in preparing them by centrifugation.
Hi,
you might wish to use another Ca2+ sensitive dye that decreases fluorescence rather then increases fluorescence upon Ca2+ release and calculate the ratio. This takes away all differences in loading inbetween individual samples.
Here is my protocol for loading the dyes:
Loading with FLUO-3 AM: 10 mM stock (Fluo-4 works as well and is actually brighter)
Prepare: 2.5 ml RPMI (without any additives)
1.7 mikroL stock FLUO-3 (7 mikroM)
25 mikroL rat serum
7.5 mikroL FURA-Red stock (+ AB`s after need; but be careful AB`s might interfere or change Ca2+ fluxes; 35 mikroM)
resuspend cells carefully and place at 37°C for 40 min.
Good luck. MVH Knut
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