Fura-2 Ca2+ Imaging of Cellular Influx

Changes of intracellular Ca²⁺ were measured using ratio Ca²⁺ imaging as we describe previously. Pre-warmed DMEM/F12 medium was used to dilute Fura-2 AM (Thermal Fisher Scientific, F1221) to a working concentration at 2.5 μM, and 0.02% Pluronic™ F-127 (Thermal Fisher Scientific, P3000MP) was added to facilitate loading of Fura-2 AM. Ca²⁺ influx was measured by perfusing the cells with Tyrode’s solution under different treatments. Ionomycin (Iono) at 1 μM was applied at the end of the experiment as an internal control. Fluorescence intensities at 510 nm with 340 nm and 380 nm excitation were collected at a rate of 1 Hz using CoolSNAP HQ2 (Photometrics) and data were analyzed using NIS-Elements (Nikon). Ca²⁺ influx induced by oxLDL or TSP1 was normalized to the maximal response caused by ionomycin as previously performed ^{57 (link)}.