Influx of calcium ions measured by flow cytometry.
A: The influx of Ca2+ was stimulated by short-circuiting via the droplets (3.0 kV, 1 or 5 shorts). A 3.0 μl droplet containing 1.0×105 Fluo-4-loaded Jurkat cells suspended in HEPES-buffered saline with (+) or without (-) 10 mM CaCl2 was added to the silicone oil, and short-circuiting via a droplet was performed. (A-1) Typical flow cytometry histograms. The fluorescence intensity was determined for Fluo-4. The flow cytometry histogram of the untreated control is filled gray. The black dashed line indicates the threshold of the Fluo-4 positive cells. The percentage of the Fluo-4 positive cells is shown. Results are representative of at least three independent experiments. (A-2) The population of Fluo-4 positive cells, as determined using flow cytometry. Data are expressed as the mean ± SD of at least three measurements. Statistical significance was determined using Student’s t-tests, *p < 0.05, **p < 0.01, ***p < 0.001 vs. ctrl. B: Droplet bouncing (6.56 kV/cm, 10 minutes) did not increase Fluo-4 fluorescence intensity. (B-1) Typical flow cytometry histograms. A 3.0 μl droplet containing 1.0×105 Fluo-4-loaded Jurkat cells suspended in HEPES-buffered saline with 10 mM CaCl2 was added to the silicone oil, and droplet boncing was performed. The flow cytometry histogram of the untreated control is filled gray. The result is representative of four independent experiments. (B-2) Median Fluo-4 fluorescence intensity, as determined using flow cytometry. Data are expressed as the mean ± SD of at least three measurements.
