Measuring Intracellular Calcium Dynamics in HUVECs

[Ca²⁺]_i measurements were performed as previously described (20 (link)). Briefly, HUVECs were seeded on round glass cover slips placed in 12-well plates and treated with PTH (100 pM) for 24 h before measurement. The cells were incubated for 20 min at 37°C with 2 μM Fluo-8/AM and 0.02% pluronic F-127 (Invitrogen) in the culture media. Intracellular Ca²⁺ stores were depleted using 2 μM TG in a Ca²⁺-free saline solution (OPSS, 140 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 10 mM glucose, and 5 mM HEPES, pH 7.3 to 7.4 adjusted with NaOH). Application of 2 mM Ca²⁺ to the medium evoked Ca²⁺ influx. The [Ca²⁺]_i is shown as fluorescence signals and was measured using a fluorescence microscope (Nikon T200, Tokyo, Japan). The baseline before the application of extracellular Ca²⁺ was considered F0, and [Ca²⁺]_i changes are expressed as the ratio of fluorescence relative to the intensity at baseline (i.e., F1/F0).