Verapamil hydrochloride

The following drugs and solvent were purchased from Sigma-Aldrich: buprenorphine hydrochloride (B9275, USDEA C-III), (±)-methadone hydrochloride (M0267, USDEA C-II), naltrexone hydrochloride (N3136), and DMSO (D8418). Naloxone hydrochloride (0599), (±)-verapamil hydrochloride (0654), E-4031 dihydrochloride (1808), and tetrodotoxin citrate or TTX (1069) were purchased from Tocris Bioscience. Norbuprenorphine hydrochloride was purchased from Noramco. ATX-II (STA-700) was purchased from Alomone Labs. To make stock solutions for patch clamp experiments, TTX, verapamil, E-4031, ATX-II, naloxone, and naltrexone were dissolved in water. Buprenorphine, norbuprenorphine, and methadone were dissolved in DMSO. When DMSO was used as a solvent to prepare stock solution, the final DMSO concentration applied to overexpression cells was ≤0.3% for Ca_V1.2 cells and ≤0.1% for the rest. In iPSC-CM experiments, stock solutions were all made with DMSO, and the final DMSO concentration applied to the cells was ≤0.1%. Aliquoted stock solutions were stored at -20°C until the day of experimentation and were thawed, vortexed, and diluted to specific test concentrations in extracellular solution.

Picrotoxin (Abcam, Cambridge, UK), bemegride (Cedarlane, Burlington, ON, Canada), diazapem (Tocris Bioscience, Bristol, UK), and thiocolchicoside (Cedarlane, Burlington, ON, Canada) were first dissolved in DMSO for the stock solutions (100 mM) and further diluted in ECS to the desired working concentration during the experiment. Bicuculline (Hellobio Inc, Princeton, NJ, USA) and flumazenil (Tocris Bioscience, Bristol, UK) were first dissolved in DMSO for the stock solutions (20 mM) and further diluted in ECS during the experiment. Verapamil hydrochloride (Tocris Bioscience, Bristol, UK) was diluted in water to make a 4 mM stock solution.

The side population (SP) of LSK cells was analyzed by FACS according to the method reported by Goodell^{53 (link)}. Total bone marrow cells were stained with Hoechst33342 dye (Sigma-Aldrich Japan K.K., Tokyo, Japan) for exactly 90 min at 37 °C. Hoechest33342-positive cells were incubated with verapamil hydrochloride (Tocris bioscience, Bristol, UK) for gate setting, and then mononuclear cells were separated by Ficoll-Paque Plus. Next, these cells were incubated with PE-Cy7-conjugated streptavidin, APC-conjugated anti-c-kit, APC-Cy7-conjugated anti-Ly6A/E (Sca-1) and PE-conjugated anti-TNF-α or FITC-conjugated anti-CD106 after Biotin Mouse Lineage Panel staining. Finally, for dead cell depletion, propidium iodide (Sigma-Aldrich) was added to the samples to remove dead cells before analysis using a FACS Aria Fusion with FACS DIVA software (BD Biosciences).