Fura-2 Calcium Imaging Protocol

Cells were seeded on gelatin-coated glass coverslips at a density of 5000 cells/cm² at 24 h before the experiments. Cells were starved in DMEM 5% FBS for at least 2 h before the experiments. Cells were next loaded (45 s at 37 °C) with 2 μM Fura-2 AM, for ratiometric cytosolic calcium concentration ([Ca²⁺]_i) measurements. During experiments, cells were maintained in standard extracellular solution of the following composition: 154 mM NaCl, 4 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 5 mM HEPES, 5.5 mM glucose. Solution pH was adjusted to 7.35 using NaOH. 4 h before experiments cells were starved in DMEM 2% FBS. Fluorescence measurements were made using a Polychrome V spectrofluorimeter (TILL Photonics, Munich, Germany) attached to an Olympus ×51 microscope (Olympus, Tokyo, Japan) and Metafluor Imaging System (Molecular Devices, Sunnyvale, CA, USA). [Ca²⁺]_i was measured using ratiometric probe Fura-2-AM and quantified according to Fiorio Pla et al. [50 (link)].