Ratiometric intracellular Ca2+measurements
Fura-2AM (Thermo Fisher, USA) was used to measure intracellular Ca2+ in real time. The cells were grown in an 8-well µ-slide (Ibidi, Germany). Mediators were contained in all incubation steps and during the measurement. A mix of 1 µM Fura-2AM and 0.02 % Pluronic (Thermo Fisher, USA) was applied for 20 min in measurement buffer (140 mM NaCl, 3.6 mM KCl, 2.6 mM CaCl2(H2O)2, 0.5 mM MgSO4, 0.5 mM NaH2PO4(H2O)2, 2 mM NaHCO3, 5 mM HEPES and 5 mM D+Glucose, pH 7.35) at 37 °C. The cells were washed twice with measurement buffer. Measuerments were performed using MetaFluor (Moleculardevices, USA) on an Axio Observer A1 (Zeiss, Germany) with a Polychrome V (Till Photonics, Germany), a CoolSNAP-Hq2 digital camera (Photometrics, USA) and a Fura-2 filter set.