Dear community!
I am about to measure cytosolic Calcium of Vero cells treated with Staurosporin or ionomycin as ionophore. In all of my experiments my Control (untreated cells) was already really high ~ 40-50000 FU and there was a high well to well variability. I can not measure significant differences in the cells treated or untreated, but the treated cells should show increased cytosolic calcium levels.
My setup is as follows:
1) Cell Seeding to 2 *10^4 overnight at 37°C, 5% CO2
2) Wash with buffer (135 mM NaCl, 4 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 5 mM glucose, 10 mM HEPES)
3) Add Staurosporin (600 nM) or ionomycin (2 mM) in 100 µL of medium and incubate for defined time steps
4) Wash with buffer (135 mM NaCl, 4 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 5 mM glucose, 10 mM HEPES)
5) Add 100 µL of Fluo-4 Direct (with probenicid, diluted in assay buffer)
6) Mesure at 495 nm excitation source and a 516 nm emission
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