Fluo 4 am

Fully differentiated iWAT cells with the indicated genotype were seeded at 40,000 cells per well in collagen I-coated 96-well black clear-bottom plates and incubated overnight. Intracellular calcium concentration ([Ca]_i) was monitored with the [Ca]_i indicator Fluo-4 AM (F14201, ThermoFisher). Cells were loaded with 5 μM Fluo-4 AM and 0.02% Pluronic F-127 (P3000MP, ThermoFisher) for 30 min at 37 °C in Hank's balanced salt solution (HBSS). Cells were then washed twice and incubated in HBSS for 30 min at room temperature. Before fluorescence recording, the buffer was changed with calcium-free HBSS. Following baseline recording (F₀), 2 μM thapsigargin or 50 μM 2-APB was added at the indicated time-point and then the fluorescence was recorded. Fluorescence signals were measured with a Spectramax M5 multimode plate reader (Molecular Devices; excitation, 494 nm; emission, 516 nm).

All chemicals were obtained from Sigma-Aldrich (Poole, UK) except fluo4-AM (acetoxymethylester), which was from Thermo Fisher Scientific, Swindon, UK. As the observed effect of SKF was unexpected, we tested the drug from four different suppliers [Calbiochem/Merck (UK), Tocris (Abingdon, UK), Abcam (Cambridge, UK), and Sigma-Aldrich]. Similar results were obtained in every case. fluo4-AM was prepared in dimethylsulphoxide (DMSO) containing 20% Pluronic F-127 (Thermo Fisher). P4 and RU1968 were dissolved in DMSO at 10 mM and diluted in supplemented Earle’s balanced salt solution (sEBBS) prior to use. The concentration of DMSO in imaging experiments was 0.03–0.3%. RU1968 was a kind gift of Dr Timo Strünker, Centre of Reproductive Medicine and Andrology, Münster, Germany.

The cytosolic Ca²⁺ concentration was evaluated using the fluorescent Ca²⁺ indicator Fluo-4/AM (Thermo Fisher Scientific). Briefly, 1 × 10⁴ cells/well of HDFs were seeded in clear flat-bottom black 96-well culture plates with the complete medium overnight. The cells were treated with ZLE or tricin at the indicated concentrations in the complete medium for 24 h. Then, the medium was changed to Hanks Balanced Salt Solution (HBSS, Thermo Fisher Scientific) supplemented with 2 μM Fluo-4/AM and incubated for 30 min at 37 °C. The cells were washed with HBSS three times, and 200 μL PBS containing 0.9 mM CaCl₂ was added. After treatment with 20 J/cm² UVA irradiation, the cytosolic Ca²⁺ concentration was then evaluated using a microplate reader (FLUOstar Omega). The excitation and emission wavelengths were 490 nm and 525 nm, respectively. All measurements were performed in triplicate at least three times independently.

T cells were labeled with the calcium indicator Fluo-4 AM (Thermo Fisher). A total of 10⁶ cells in 1 mL of RPMI 1640 medium containing 10% FBS were loaded with 5 mM Fluo-4 AM for 20 min at 37°C in the presence of 0.1% Pluronic F-127 and 0.25 mM Probenecid (Thermo Fisher). The cells were washed three times with RPMI 1640 and incubated at 37°C for an additional 5 min. The cells were then resuspended in RPMI 1640 medium containing 10% FBS, and their Ca²⁺ levels were assessed using a CytoFLEX Flow Cytometer (Beckman Coulter). For live-cell microscopy, loaded T cells were plated on poly-L-lysine-coated culture plates, centrifuged at 100 g for 2 min, and placed in an incubator at 37°C for 30 min. Images were acquired using a BioTek-lionheart FX (BioTek).

αS species were added to the CM of SH-SY5Y cells and primary rat cortical neurons seeded on glass coverslips for 15 min at various concentrations (0.03, 0.1, 0.3, 1.0, and 3.0 μM). In a set of experiments, 0.3 µM OB* were added to the CM of SH-SY5Y cells for 0, 5, 10, 15, 30, 60, and 180 min and to primary rat cortical neurons for 0, 5, 15, 60, and 180 min. Cells were also treated with 0.3 µM OB* in CM without Ca²⁺. In a set of experiments, cells were treated for 15 or 180 min with 0.3 µM M in the absence or presence of 0.03 µM SF (monomers equivalents, corresponding to 10% of monomers). Then the cells were loaded with 10 μM Fluo-4 AM (Thermo Fisher Scientific) and the analysis was performed by confocal microscopy (excitation at 488 nm)^{37 (link),57 (link)}.
In a set of experiments, the Ca²⁺ influx was analyzed in real-time in living SH-SY5Y cells loaded with the Fluo-4 AM probe for 10 min. The intracellular Ca²⁺ basal level in living cells was measured for 10 min and then Ca²⁺ currents were analyzed following the addition of OB* or SF up to 30 min. The emitted fluorescence was detected at 488 nm excitation line over time by the confocal scanning system described above.

Intracellular calcium of different cell populations was measured in whole blood samples using Fluo-4-AM, cell permeant (Fluo-4-AM, ThermoFisher scientific, F14201). Suspended cells were incubated with Fluo-4-AM (10 μM) in the presence of pluronic F-127 (0.02%) for 15 min followed by addition of combinations of monoclonal antibody; CD-42b-PE (Platelets) in HEPES buffer for another 15 min at room temperature in the dark. Cells were then washed with HEPES buffer and resuspended in 300 μL of HEPES buffer and incubated for 20 min. A total of 20,000 events were recorded and analyzed using CytExpert program.