Fluo 4 direct ca2 assay kit

Calcium (Ca²⁺) levels were determined using the Fluo-4 Direct Ca²⁺ assay kit (ThermoFisher Scientific) and Rhod-2 calcium indicator (ThermoFisher Scientific), as previously described [14 (link)].

Ca²⁺ measurements was performed according to manufacturer’s instructions using the Fluo-4 Direct Ca²⁺ assay kit (Thermo Fisher Scientific). In brief, HUVECs were seeded in 96-well plates and Fluo-4 direct Ca²⁺ reagent containing 1X Fluo-4 direct Ca²⁺ assay buffer and 1 mM probenecid was added for 1 hour at 37ºC in 5% CO₂. When using U73122 or MAPTAM, drugs were added after 30 minutes at a concentration of 2.5 μM and 5 μM, respectively, and incubation proceeded for another 30 minutes. Fluorescence was measured at 494 nm (excitation) and 516 nm (emission), following stimulation with 100 ng/mL VEGFA or 1μm TG.