Fura 2 am

Fura-2AM (ThermoFisher) was used to determine the intracellular Ca²⁺ concentration in real time. Cells were therefore grown in an 8-Well μ-slide (Ibidi, Martinsried, Germany) until confluency. Mediators were used before the Fura-2AM dye was applied and were also present during dye loading. A mix of 1 μM Fura-2AM and 0.02% Pluronic (ThermoFisher) was applied for 20 min in measurement buffer (140 mM NaCl, 3.6 mM KCl, 2.6 mM CaCl₂(H₂O)₂, 0.5 mM NaH₂PO₄(H₂O)₂, 2 mM NaHCO₃, HEPES and 5 mM D+ Glucose) at 37°C to facilitate transfer of the dye into cells. After replacing the solution with fresh measurement buffer cells were measured with MetaFluor (Moleculardevices, San Jóse, USA) on an Axio Observer A1 (Zeiss, Jena, Germany) with a Polychrome V (Till Photonics, Planegg, Germany), a CoolSNAP-Hq2 digital camera (Photometrics, Tucson, USA) and a Fura-2 filter set.

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, DE). Mediators (Table S2) were contained in all incubation steps and during the measurement. A mix of 1 µmol/l Fura-2AM and 0.02 % pluronic (Thermo Fisher, USA) was applied for 20 min in measurement buffer (140 mmol/l NaCl, 3.6 mmol/l KCl, 2.6 mmol/l CaCl₂(H₂O)₂, 0.5 mmol/l MgSO₄, 0.5 mmol/l NaH₂PO₄(H₂O)₂, 2 mmol/l NaHCO₃, 5 mmol/l HEPES and 5 mmol/l D+Glucose, pH 7.35) at 37°C. The cells were washed twice with measurement buffer. Measurements were performed using MetaFluor (Moleculardevices, USA) on an Axio Observer A1 (Zeiss, DE) with a Polychrome V (Till Photonics, DE), a CoolSNAP-Hq2 digital camera (Photometrics, USA) and a Fura-2 filter set. For each independent experiment, the signals from 8 out of 15 randomly selected cells were evaluated (occasional non-responding cells were not included, very rare oscillating cells and weak responders were included, N = 4).

[Ca²⁺]_i measurements in HEK293 cells were carried out using the fluorescence indicator fura-2-acytoxymethyl ester (Fura 2-AM; ThermoFisherScientific, Darmstadt, Germany; #F1201) combined with a monochromator-based imaging system (T.I.L.L. Photonics; FEI, Gräfeling, Germany) attached to a fluid immersion objective (LUMPLFLN 40XW/0.80 w). Cells were loaded with a cocktail composed of 2.5 μM Fura 2-AM, 0.01% pluronic-F127 (ThermoFisherScientific, Darmstadt, Germany; #P6866) for 30 min at room temperature (22–24 °C) in a standard Hank’s Balanced Salt Solution (HBSS) buffer composed of 138 mM NaCl, 6 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂ and 10 mM HEPES ([4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid]) adjusted to pH 7.4 with NaOH at room temperature. Afterward, cells were washed with HBSS and left for another 30 min at RT in HBSS. Cover slips were then mounted in a bath chamber made of plexiglas on the stage of the microscope (Olympus BX51WI, Hamburg, Germany). Ca²⁺ influx was recorded and visualized in TillVision Live Acquisition and Offline Analysis software [formerly FEI Munich GmbH (Till Photonics), now Thermo Fisher Scientific] as a ratio of 340/380 nm with a 40× objective. Ca²⁺-bound Fura2-AM is excitable at 340 nm and the unbound state of Fura2-AM at 380 nm. The ratio was calculated by analyses of emission which was detectable at 510 nm after excitation with each wavelength.

The cells, treated or not, were incubated in the Fura-2AM loading solution consisted of standard extracellular saline (SES; 10 mM HEPES pH 7.4, 135 mM NaCl, 5mM KCl, 1.2 mM MgCl₂, 2 mM CaCl₂, 1 mM bicarbonate and 5 mM glucose) with 0.1% BSA and 10 μM Fura-2AM (Thermo Fischer Scientific) at 37°C for 30 min. The loading solution was then removed and the cells were equilibrated in fresh SES buffer for 15 min and detached using trypsin. The fluorescence of the cell suspensions (1 mL) was recorded using a SFM 25 (Kontron Instruments; excitation wavelengths: 340 and 380 nm, emission wavelength: 510 nm). The changes in the intracellular calcium concentration were monitored using the Fura-2 340/380 fluorescence ratio.

For the measurement of Ca²⁺ levels, fura-2, AM (Invitrogen, 2256798, USA) was used. After 24 h, PDLCs were washed by PBS for three times and then stained with 2 μM fura-2-AM for 45 min at 37°C and measured in a microplate reader (Thermo Fisher Scientific, USA).