Seahorse xf dmem assay medium ph 7

The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of 5 ng/mL TGF-β2- or 250 nM DEX-treated or untreated 2D cultured HTM cells (as above) and those treated with 10 μM pan-ROCK-i ripasudil, and ROCK2-i KD025 for 24 h were each measured using a Seahorse XFe96 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s instructions. In brief, 20 × 10³ 2D cultured cells per well were placed in a XFe96 Cell Culture Microplate (Agilent Technologies, #103794-100). After centrifugation of the plate at 1600 g for 10 min, the culture medium was replaced with 180 μL of the assay buffer (Seahorse XF DMEM assay medium (pH 7.4, Agilent Technologies, #103575-100) supplemented with 5.5 mM glucose, 2.0 mM glutamine, and 1.0 mM sodium pyruvate). The assay plates were incubated in CO₂-free incubator at 37 °C for 1 h prior to the measurement. OCR and ECAR were measured in the Seahorse XFe96 Bioanalyzer under the 3 min mix and 3 min measure protocols at baseline and following injections of oligomycin (final concentration: 2.0 μM), carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP, final concentration: 5.0 μM), a mixture of rotenone/antimycin A (final concentration: 1.0 μM), and 2-deoxyglucose (2-DG, final concentration: 10 mM).