Gen5 version 2

Previous studies have shown that C2C12 cells represent a model to study insulin resistance in diabetes and improvement in muscle function [21 (link), 22 (link)]. C2C12 skeletal muscle cells (American Type Culture Collection) were maintained in DMEM with 10% FBS and antibiotics, at 37°C in a humidified incubator of 5% CO₂. When the cells reached 80% confluence, C2C12 cells were differentiated into skeletal myotubes in DMEM-low glucose with 2% horse serum for 5 days. Viability levels of C2C12 cells were determined by the ability of mitochondria to convert MTT to an insoluble formazan product. Cells were maintained in 96-well plates at a density of 3 × 10⁴ cells/well and subsequently subjected to different concentrations of DPHC (0.1, 2, 10, and 30 μM) for 24 h. Cells were pretreated with MTT solution (2 mg/mL) for 3 h. Subsequently, cell density was determined by measuring optical density (OD) at 540 nm using a microplate reader (Gen5 version 2.05, BioTek, Winooski, Vermont, USA).

Using 0.1 mM ATP standard dilution, a standard curve was generated following the ATP assay kit protocol (ab83355, Abcam, America). 10 mg of muscle tissue was harvested for each assay and washed in cold PBS. Tissue was homogenized in ice-cold 2 N PCA with 10-15 passes. Following maintaining on ice for 30-45 min, samples were centrifuged at 13,000 g for 3 min at 4°C, and supernatants were collected. Samples were adjusted to pH between 6.5 and 8 to neutralize the samples and precipitate the excess PCA. Samples were centrifuged at 13,000 g for 15 min at 4°C, and supernatants were collected. The supernatants were transferred to individual wells of 96-well plates mixed with the same volume of ATP reaction mix and incubated at 21 ± 1°C for 30 min, protected from light. ATP levels in each sample were determined by measuring optical density (OD) at 570 nm using a microplate reader (Gen5 version 2.05, BioTek, Winooski, Vermont, USA). The amount of ATP in the samples was calculated according to the standard curve.

Intracellular ROS were quantified by the 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCF-DA Cat. No. D6883; Sigma). C2C12 cells were plated (2000 cells/well) into special optics 96-well plates (Corning-Costar, New York, NY, USA) and the cells were treated with RSV at 1 µM and Compound 6 at 1 and 10 µM for 24 h. After the incubation, cells were washed with an imaging buffer and treated with 300 µM H₂O₂ for an immediate fluorescence measurement. Plates were read every 50 s from 0 to 10 min for kinetic data analysis using a microplate reader (Synergy H1, BioTek), with excitation and emission wavelengths at 490 nm and 520 nm, respectively, and analyzed by Gen 5 version 2.08 (BioTek) [56 (link)].

SHSY-5Y human neuroblastoma cells were plated (2700 cells/well) into special optics 96-well plates (Corning-Costar, New York, USA) and the cells were treated with retinoic acid (RA)/phorbol 12-myristate 13-acetate (PMA) in order to obtain a dopaminergic phenotype. The differentiated cells were treated with SP1–6 at 1 µM for 24 h and then washed with an imaging buffer (125 mM NaCl, 5 mM KCl, 1.2 mM MgSO₄, 5 mM glucose, 25 mM HEPES, and 2 mM CaCl₂). A concentration of 10 µM of 2′,7′-dichlorofluorescin diacetate (H₂DCFDA, Molecular probes, Life Technologies REF D399) was added and the plates were incubated at 37 °C for 30 min. After the incubation, cells were washed with an imaging buffer and treated with 25 µM H₂O₂ for an immediate fluorescence measurement.
Plates were read every 50 s from 0 to 5 min for kinetic data analysis using a microplate reader (Synergy H1, BioTek), with excitation and emission wavelengths at 490 nm and 520 nm, respectively, and analyzed by Gen 5 version 2.08 (BioTek).