Cal 520

The PSS consisted of (in mM):145 NaCl, 2.0 MOPS, 4.7 KCl, 1.3 NaH₂PO₄, 5.0 Glucose, 1.17, MgCl, 2.0 CaCl, 0.02 EDTA (pH adjusted to 7.4 with NaOH). In experiments using Ca²⁺ free PSS, CaCl₂ was replaced with MgCl₂ on an equimolar basis and EGTA (1 mM) was included. Caged-IP₃ (caged-IP₃ 4,5-dimethoxy-2-nitrobenzyl) was obtained from Sichem (Germany). Cal-520 was obtained from Abcam (UK). Pluronic F-127 was obtained from Invitrogen (UK). Mitotracker Green FM was obtained from Invitrogen (UK). All other drugs and chemicals were obtained from Sigma (UK). Stock solutions of ACh, catalase-polyethylene glycol and H₂O₂ were prepared by dissolving each chemical in double-distilled, dionized water. 2-aminoethoxydiphenyl borate (2-APB), caged-IP₃, Cal-520, carbonyl cyanide m-chlorophynyl hydrazine (CCCP), oligomycin, TMRE and Mitotracker Green FM were dissolved in DMSO.

In black 96-well plates, RASF were incubated with 4 µM of calcium dye Cal-520 (ab171868, Abcam, Cambridge, UK) in PBS with 0.02% Pluoronic F127 (Thermo Fisher scientific, Waltham, MA, USA, # P6866) for 60 min at 37 °C, followed by 30 min at room temperature. After washing, HBSS or PBS containing 1 µM PoPo3 iodide (Thermo Fisher scientific, # P3584) and respective antagonists/ligands/inhibitors were added for 30 min at room temperature. After that, THC was added and the intracellular Ca²⁺ concentration as well as PoPo3 uptake were evaluated with a TECAN multimode reader over 90 min.

For quantification, events were measured on a LSR II Flow Cytometer unless otherwise specified. NBD-lipid (Avanti Polar Lipids), FITC-Annexin V (Biolegend), Cal520 (Abcam), and FITC (Sigma) fluorescence were detected at 488 nm ex/ 530 nm em. Hoechst 33342 (Thermo Fisher) fluorescence was detected at 410 nm ex/ 450 nm em. PerCP-anti-CD14 (Miltenyi Biotec) was detected at 488 ex/ 670 nm em. Data were initially processed using FlowJo. RBCs, saponin-isolated parasites, or monocytes were gated on FSC and SCC. iRBCs (positive) and uRBCs (negative) were differentiated by Hoechst fluorescence. Saponin-isolated parasites were gated on positive Hoechst fluorescence, and monocytes were gated on positive Hoechst and PerCP-anti-CD14 fluorescence. The geometric mean of fluorescence intensity (mean fluorescence intensity, MFI) and/or the percentage of each population (e.g. FITC positive) was calculated with FlowJo. Background fluorescence was subtracted based on unstained controls (buffer or solvent only). Except where otherwise specified, MFI data was normalised between experiments by setting the fluorescence of untreated uRBCs to 1 or 100%.

In black 96-well plates, RASF were incubated with 4 µM of calcium dye Cal-520 (ab171868, abcam, Cambridge, UK) in Hanks buffered salt solution (1 mM Ca²⁺; HBSS, sigma, # 55037 C) or PBS (no Ca²⁺) with 0.02% Pluoronic F127 (Thermo fisher scientific, Waltham, USA, # P6866) for 60 min at 37 °C followed by 30 min at room temperature. After washing, HBSS or PBS containing 1 µM PoPo3 iodide (Thermo fisher scientific, # P3584) and respective antagonists/ligands/inhibitors were added for 30 min at room temperature. After that, CBD was added and the intracellular Ca²⁺ concentration as well as PoPo3 uptake were evaluated with a TECAN multimode reader over 90 min.

The 1 × 10⁴ cells were seeded in black flat 96-well plates (Greiner Bio-One, Kremsmünster, Austria). For measurement of intracellular calcium concentration Cal-520 No Wash Calcium Assay (Abcam, Cambridge, UK) was used. Five µM of the fluorescent dye Cal-520 (Abcam, Cambridge, UK) were added to Hank’s Balanced salt solution (HBSS, Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (LONZA, Basel, Switzerland), 0.02% Pluronic^TM F-127 (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) and 1 mM Probenecid (Sigma-Aldrich St. Louis, MO, USA) from which 100 µL were given to each well. Afterward, incubating the plate for 90 min at 33 °C or 39 °C and adding 100 µL Probenecid dissolved in Minimum Essential Medium (MEM, Gibco, Thermo Fisher Scientific, Waltham, MA, USA) intracellular calcium concentration was measured at Ex/Em = 490/525 nm with Tecan infinite F2000 Pro (Tecan, Männedorf, Switzerland).

Ca²⁺ transient analysis was performed to evaluate the Ca²⁺ handling function between CTRL, AA-, and GW-treated hiPSC-CMs monolayers. Briefly, cells were incubated 30 min in FluoroBrite DMEM Media (Thermo Fisher) supplemented with 1.25 µM Cal-520 (Abcam) and 0.02% Pluronic F-127 solution (Sigma-Aldrich). 30 s at 33 frames per second (fps) videos were automatically scanned by a Leica Thunder microscope. Analysis was conducted using Cyteseer (Vala Sciences, California, USA) as previously described [50 (link)]. The physiological parameters: rise time, calcium transient duration (CTD) at 25 and 75 percent, full width at half maximum (FWHM) representing 50% of the peak width, decay time, and beats per minute were automatically calculated for each time series. Data tables were analysed with Microsoft Excel and drug responses and bar plots were generated with GraphPad Prism 9 software.
Videos of monolayers of iPSC-CMs were taken using a GoPro Black Hero 7 camera connected to a bright field microscope via c-mount system. A 20 s video was taken at endpoint (D27), and beating rate (beats per minute, bpm) was manually calculated.