Fluoforte

T84 cells were seeded at 1×10⁵ per transwell on the underside of transwells [88 (link)] and cultured for ~10 days until TEER reached >1000 Ω. Cells were pre-treated with or without the Ca²⁺ inhibitors, 2APB (10 μM, Sigma, Saint Louis, MO) and BAPTA (50 μM, Sigma), for 1 h and incubated with GC (MOI of 10) apically in the presence or absence of the inhibitors for 4 h. Then cells were incubated with the fluorescent Ca²⁺ indicator Fluoforte (100 μg/ml, Enzo Life Sciences, Farmingdale, NY) or Fluo-4 (100 μM, Life Technologies) for 1 h. Confocal xz images were acquired in the presence of the membrane dye CellMask (5 mg/ml, Life Technology) using Leica TCS SP5X confocal microscope (Leica Microsystems, Buffalo Grove, IL), based on the instruction by manufacturers. To quantify the intracellular Ca²⁺ level, the cytoplasmic region of individual cells was manually selected based on the CellMask staining in randomly acquired confocal images, and the mean fluorescent intensity (MFI) of Fluoforte and Fluo-4 in the cytoplasmic region was measured using the NIH ImageJ software.

A perforated patch whole-cell current clamp was used to measure membrane potential, as previously described (15) (link). [Ca²⁺] variations were simultaneously monitored using the Ca²⁺-sensitive fluorescent dye FluoForte (Enzo Life Sciences, Farmingdale, New York). A whole-cell ruptured patch voltage clamp was used to record peak and late Na⁺ current, L-type Ca²⁺ current (I_Ca-L), and delayed rectifier K⁺ current (I_K), using appropriate protocols and solutions (15) (link).

To determine changes in the intracellular Ca²⁺ concentration, 2 × 10⁴ PDAC cells were plated onto 96-well black microplates with clear bottoms and grown overnight. After treatment with SLURP1, nicotine, the specific CHRNA7 antagonist MLA, or the combinations, cells were loaded with FluoForte^® (FluoForte^® Calcium Assay Kit; Enzo^® Life Science, Farmingdale, NY, USA) which had been dissolved in Hank’s buffer with a dye efflux inhibitor. The kinetic measurement of fluorescence was performed using a Synergy^TM HTX Multi-Mode microplate reader (BioTek, Winooski, VT, USA).

For Fluoforte and Rhod‐2 calcium assays, 6 days after siRNA transfection in glass‐bottom dishes (Thermo Scientific), live cells were treated either with 5 µM Fluoforte (ENZ‐52014, Enzo Life Sciences) or with 10 µM Rhod‐2 (R‐1245MP, Thermo Scientific) for 1 hr at 37 °C in Hank’s balanced salt solution (HBSS) with calcium, magnesium, and no phenol red (14025050, Thermo Scientific). Cells were then washed and incubated in HBSS with no calcium, no magnesium, and no phenol red (14175053, Thermo Scientific) for calcium imaging. Fluorescence was recorded every 1.5 s using Zeiss LSM 780 confocal microscope. Histamine (H7125‐1G, Sigma‐Aldrich) was injected after 1 min of measurement at a final concentration of 100 nM. Average changes in fluorescence intensities in multiple regions of interest (ROI) were calculated. Results are shown as (F1‐F0)/F0, where F0 is the mean of the intensities from 10 to 50 s. Alternatively, cells stably expressing mitoGCaMP2, a genetically encoded mitochondrially targeted calcium indicator, were transfected with siRNA in glass‐bottom dishes. Six days later, fluorescence (Ex 488 nm/Em 500–570 nm) was monitored in live cells using Zeiss LSM 780 confocal microscope. Fluorescence intensity was quantified using the ImageJ software.

In cardiomyocytes freshly isolated from patient ventricular tissue, we simultaneously measured membrane potential and calcium transients using perforated patch whole-cell current-clamp combined with fluorescence recordings after loading with the Ca²⁺-sensitive fluorescent dye Fluoforte (Enzo Life Sciences, Farmingdale, NY, USA), as previously described [31 (link)]: fluorescence was measured at 515 ± 10 nm during excitation at 490 ± 8 nm. For hiPSC-CMs, we used whole-cell current clamp to measure APs; the pipette solution contained (in × 10⁻³ M) 115 K methanesulfonate, 25 KCl, 10 (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES), 3 MgCl₂, and cells were perfused with Tyrode buffer containing 1.8 × 10⁻³ M CaCl₂. APs were elicited with short depolarizing current pulses (< 3 ms) at 1 Hz frequency. Action potentials were analyzed for MDP (mV), amplitude (mV), and action potential duration (ADP50 and APD90, ms) using the Clampfit 10.7 software (Molecular Devices, San Jose, CA, USA).