Intracellular Calcium Mobilization by MCP-1

Intracellular calcium mobilization was monitored using the Fluo-8 No Wash Calcium Assay Kit (Abcam) according to the manufacturer’s instructions for non-adherent cells. Healthy donor neutrophils isolated as described above were resuspended in equal amounts of HBSS supplemented with 1.26 mM CaCl₂ and 20 mM HEPES and Fluo-8 dye-loading solution. A total of 10⁵ cells/well/100 μl were seeded into sterile, black, flat-bottom 96-well plates and incubated for 30 min. Pre-warmed HBSS supplemented with 1.26 mM CaCl₂ was added to a final assay volume of 270 μl. The plate was transferred into a fluorescence plate reader with a dual injector function (Varioskan Flash, Thermo Scientific) and monitored at 37°C for 85 min reading fluorescence at an excitation wavelength of 490 nm and an emission wavelength of 525 nm. After baseline recording, MCP-1 was injected at medium speed (BioLegend, 0.5 ng/ml final concentration) and fluorescence was measured per well immediately afterward. The plate was scanned every minute for 20 min. Then, ionomycin was injected at medium speed (Sigma, 1.3 μM final concentration) and fluorescence was again measured per well immediately afterward. Calcium mobilization was monitored for 15 min every minute and then for 50 min every 5 min. Results are presented as fold change to baseline.