Hbss solution

iPSC-derived dopaminergic neurons were grown until 48 DIV and were then imaged with FM1-43 (Thermo Fisher Scientific). Cells were washed in HBSS−/− supplemented with 5 mM glucose and 10 mM HEPES. 2 μM FM1-43 was then applied for 1 min before uptake was induced with 75 mM NaCl and 10 mM KCl and left for 1 min before being washed in HBSS−/− solution (Thermo Fisher Scientific). Images were acquired at 37C in 5% CO₂ on the Opera Phenix (Perkin Elmer) at 63× magnification. Images were quantified in ImageJ by measuring the fluorescence intensity of puncta at each timepoint.

MMP was measured by using fluorescent probe JC-1 (Santa Cruz, Cat. No.: sc-364, 116). After galangin (20 μM) treatment for 24 and 48 h, MGC 803 cells were rinsed with 1×HBSS solution (Gibco, Cat. No.: 14025–092) and incubated with JC-1 (10 μM) at 37°C for another 30 min. After that, the cells were rinsed with 1 × HBSS solution once again, and the fluorescent intensity of the JC-1 monomers and aggregates was detected under different conditions (Ex (λ) 485 nm, Em (λ) 530 nm for monomers; Ex (λ) 530 nm, Em (λ) 590 nm for aggragates) on a microplate reader (Varioskan Flash, Thermo Scientific, United States). Fluorescent images were captured under a fluorescent microscope (IX81).

Isolation and culture of neonatal rat cardiomyocytes were performed as previously described56 (link). Briefly, Spraque Dawley (postnatal 1–2 days) were euthanized using isoflurane inhalation followed by cervical dislocation. Then ventricles were digested in HBSS solution containing 0.1% trypsin (Invitrogen, USA) and 0.05% type II collagenase (Worthington, USA). Cells were pre-plated for 2 h and the supernatant containing purified cardiomyocytes was collected and cultured for another 48–72 h before transfecting the adenovirus.

Samples were obtained from Left ventricular at indicated time points. Single cell suspensions were prepared as described previously, with some modifications (20 (link)). Briefly, hearts were cut into small pieces and digested with 0.3 mg/ml collagenase II (Invitrogen, USA), 0.3 mg/ml dispase II (Sigma, USA), DNase I (Biosharp, China) and 2.5 mM Cacl₂ (Mackin, china) in HBSS solution (Invitrogen, USA) for 45 min at 37°C with gentle agitation. After the digestion, primary cardiac cells were obtained using Percoll (Solarbio ^®) gradient separation and passed through 70-μm cell strainer. The obtained cells were washed with RPMI-1640 cell culture medium for further analysis.

DRGs were harvested from thoracic spinal levels of adult Pvalb^cre;Rosa26^Ai14 and Pirt^cre;Scn1a-floxed (6–16 weeks) mice of both sexes and transferred to Ca²⁺-free and Mg²⁺-free HBSS solution (Invitrogen, 14170-112). Upon isolation, processes were trimmed, and ganglia were transferred into collagenase (1.5 mg/mL; Type P, Sigma-Aldrich, 11213865001) in HBSS for 20 min at 37°C followed by TrypLE Express (Thermo Fisher, 12605-010) for 3 min with gentle rotation. TrypLE was neutralized with 10% horse serum (heat-inactivated; Invitrogen, 26050-070) and supplemented with culture media (MEM with l-glutamine, Phenol Red, without sodium pyruvate, Thermo Fisher, 11095-080), containing 10,000 U/mL penicillin-streptomycin (Thermo Fisher, 15140-122), MEM Vitamin Solution (Invitrogen, 11120-052), and B-27 supplement (Thermo Fisher, 17504-044). Serum-containing media was decanted and cells were triturated using a fire-polished Pasteur pipette in the MEM culture media described above. Cells were resuspended and triturated using a plastic pipette tip. Cells were plated on glass coverslips that had been washed in 2 M NaOH for at least 4 hr, rinsed with 70% ethanol, UV-sterilized, and treated with laminin (0.05 mg/mL, Sigma-Aldrich, L2020-1MG) for 1 hr prior to plating. Cells were then incubated at 37°C in 5% CO₂. Cells were used for electrophysiology experiments 14–36 hr post-plating.