Carbonyl cyanide p trifluoromethoxyphenylhydrazone cccp

Mitochondrial function was assessed in live cells using an XFp and XFe96 Extracellular Flux Analyzer (Agilent Technologies). A total of 1 × 10⁴ T-Rex HEK or 1.5 × 10⁵ iPSC-derived neurons were seeded in XFpSeahorse microplates and allowed to adhere overnight (for HEK cells) or for 7 days (iPSC-derived neurons). Measurements of the oxygen consumption rate (OCR) were performed in a freshly prepared assay medium, pH 7.4 (Seahorse XF DMEM), according to the manufacturer’s protocol. The OCR was measured before and after the serial addition of 20 or 1 μΜ oligomycin, 1 or 5 μΜ carbonyl cyanide p-trifluoromethoxyphenylhydrazone (CCCP), 2 or 1 μΜ antimycin A and 2 or 1 µΜ rotenone to T-Rex HEK cells or iPSC-derived neurons, respectively (all from Sigma–Aldrich). Following each injection, three measurements for a total period of 15 min were recorded. The data were analyzed using Wave 2.6 software (RRID:SCR_014526), and OCR parameters (basal respiration, maximal respiratory capacity, respiratory reserve, and ATP-linked respiration) were calculated. At least three technical replicates per condition were used, and the experimental values were normalized to the protein content per well via a BCA assay.

To analyze mitochondrial respiration, the OCR was assessed using an XFe96 extracellular flux analyzer (Agilent Technologies). The HSC-4 cells were seeded at a density of 2 × 10⁴ cells/well in Seahorse XFe96 cell culture microplates (Agilent, Santa Clara, CA, USA). After adherence and equilibration, the Chaga mushroom extract was treated with various concentrations (160, 200, 400, and 800 µg/mL) for 24 h. The OCR was then assessed by sequential injections of oligomycin (Sigma-Aldrich), carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (CCCP; Sigma-Aldrich), rotenone (Sigma-Aldrich), and antimycin A (Sigma-Aldrich), followed by incubation with XF assay medium (Agilent Technologies). All OCR results were normalized to concentrations of cellular protein per well using a Pierce™ BCA protein assay (Thermo Fisher Scientific).