Mitoplate s 1

Experiments were performed using the Biolog MitoPlate S-1 assays (Biolog, Hayward, CA, United States) using 6 × 10⁵/well NPCs and 4 × 10⁴/well astrocytes in 96 well culture dishes as described (Ryu et al., 2021 ). OD 590 was measured at various time points (0, 1, 2, 3, 4, 5, 6, 24, and 48 h) using a Synergy HT BioTek plate reader (BioTek). Measurements were normalized to and calculated as percent change from no substrate control. Each measurement was done in triplicate and cell lines were analyzed in two repeat experiments.

Bioenergetic parameters were determined using Seahorse XFp Cell Mito Stress Tests (Seahorse, Agilent Technologies, Santa Clara, CA) and the processing of bioenergetic substrates were assessed in Biolog MitoPlate S-1 assays (Biolog, Hayward, CA). To determine biochemical compounds and function, the NAD/NADH-Glo™ and the Glucose Uptake-Glo™ Assay Kits (Promega, Madison, WI) were used. Mitochondrial densities were determined with the MitoTracker^TM Green FM dye (Invitrogen Thermo Fisher Scientific, Waltham, MA).

Mitochondria function was examined using the MitoPlate S‐1 from Biolog, which measures the sensitivity of mitochondria to 22 diverse inhibitors. Human skeletal myoblast cells were expanded and then reseeded at 30,000 cells per well on the MitoPlate S‐1 according to the manufacturer's instructions. The plate was incubated with cells at 37°C and 5% CO₂ for at least 16 h and then was read on a plate reader using a 595‐wavelength filter. To examine the function of the electron transport chain a membrane sensitive JC‐1 Dye was used. Cells were plated and grown for 48 h in collagen‐coated 96‐ well dishes in growth media at 37°C and 5% CO₂. Once cells were 80% confluency the cells were rinsed with warm Phosphate Buffered Saline (PBS) and then incubated in 5 μM JC‐1 as described by Kolb et al., (Kolb et al., 2018 (link)). Following the 30‐minute incubation, the cells were rinsed with warm PBS and dye excitation was measured using Molecular Devices SpectraMax M5 microplate reader with an excitation wavelength of 488 nm. Emission was measured at both 527 and 590 nm (Kolb et al., 2018 (link); Sivandzade et al., 2019 (link)).

Mitochondrial function assays were performed using MitoPlate S-1(Biolog Inc., Hayward, CA). The substrates on MitoPlate S-1 were first dissolved by incubating the plate with 30 µl of Assay Mix, consisting of 2x BMAS, 6x Redox Dye MC and saponin (30 µg/ml) necessary for cell permeabilization in a 5% CO2 incubator at 37°C for 1 h before inoculating 400,000 cells per well in a volume of 30 µl. Color changes are read kinetically during 24h and are performed with OmniLog instrument (Biolog Inc., Hayward, CA). Signal intensity was measured at 24h and corrected by background subtraction with the negative control condition without substrate. The relative intensity was computed by dividing each intensity by the global mean of a particular sample.