Calibration solution

Rat primary neurons were cultured on 96-well (XF96) microplate at the concentration of 3 × 10⁴ cells per well (Seahorse Bioscience, Illerica, MA) and transduced with Ad-Null, Ad-Tat or Ad-U1. Forty-eight hours after transduction, neurons were subjected to OCR measurement at 37 °C in an XF96 extracellular flux analyzer (Seahorse Bioscience). The XF96 extracellular flux assay kit was calibrated using calibration solution (Seahorse Bioscience) in a non-CO₂, 37 °C incubator overnight, mitochondrial complexes were sequentially inhibited with a: 10 µM oligomycin, b: 10 µM FCCP, c: 1 µM rotenone/antimycin A. Basal and maximal OCR, ATP coupled respiration, spare capacity and proton leak were normalized to total protein and analyzed.

Fibroblasts were cultured for 10 days in DMEM containing 25 mM galactose and supplemented with 15% FBS. XF24 extracellular flux analyzer from Seahorse Biosciences was used to measure the rates of oxygen consumption. Cells were plated the previous day of experiment on the XF24 cell culture microplates (Seahorse Biosciences) at a density of 30,000 cells per well. XF24 cartridge (Seahorse Biosciences) was equilibrated with the calibration solution (Seahorse Biosciences) overnight at 37°C. XF assay medium (5 mM galactose, 2 mM Pyruvate) in XF base media (Seahorse Biosciences) was prepared and pH adjusted to 7.0 on the day of the experiment. Oxygen consumption rates (OCRs) were measure for 3 min with 3 min of mixing and 2 min waiting period after each injection of individual cellular stress reagents in the order listed here: 500 nM Oligomycin, 500 nM FCCP, 100 nM Antimycin, and 100 nM Rotenone (Sigma–Aldrich). After the assay was completed, cells in each well were counted using ViCell cell viability analyzer (Beckman Coulter) and the counts were used to normalize the OCRs.