A7506

The following reagents were added to in vitro culture from days 2.5 to 4: methyl pyruvate (10 mM; 371173; Sigma), methyl malate (5 mM; 355-17971; Wako), etomoxir (90 μM; E1905; Sigma), rotenone (120 nM; R8875; Sigma), metformin (1 mM; 150959; SIGMA), oligomycin (1 nM; O4876; Sigma), 2-deoxy-D-glucose (200 μM; 154-17-6; SIGMA), haemin (30 μM; H9039; Sigma), ascorbic acid (200 μM; A7506; Sigma), MitoTEMPO (64 μM; ALX-430-150; Enzo Life Science) and TPP (64 μM; 309567; SIGMA); from days 1 to 4: LY294002 (3 μM; 1667; Biovision), AZD5363 (5 μM; S8019; Selleckchem) and 5-ALA hydrochloride (450 μM; 3785; Sigma); and from days 3 to 4: cobalt (II) chloride (150 μM; 232696; Sigma).
For OCR and ECAR assay, all the reagents were added at 36 h of culturing and incubated for 12 h. Exposing duration of cells to reagents was therefore from 36 to 48 h of culturing.

The following method was adapted from Gaisawat et al., (2019) [69 (link)] and Benzie and Strain (1996) [73 (link)]. A 1 mM stock solution of ascorbic acid (Sigma-Aldrich, A7506, St. Louis, MO, USA) was made and subsequent dilutions completed to obtain a standard curve. A 96-well microplate was used, where 10 µL of either sample or standard was pipetted into a well, along with 30 µL of deionized and 200 µL of a previously made FRAP solution (acetate buffer, 2,4,6-tri(2-pyridyl)-s-triazine and ferric chloride solution combined in a ratio of 10:1:1). The samples and standards were mixed by pipetting for 10 s and then incubated at RT for 8 min. The absorbance was measured at 593 nm using a µQuant microplate reader (BioTek Instruments, Winooski, VT, USA). The antioxidant capacity of the samples was calculated using an external calibration curve, where the linearity of the curve was assessed using R².