Xf 24 metabolic flux analyzer

The OCR rate of in vitro grown MCA205 cells in vitro was measured using a Seahorse XF-24 Metabolic Flux Analyzer. 1 × 10⁵ cells were seeded on XF-24 V7 multi-well plates, and then treated or not with 100 μM Pant for 16 h at 37°C. The OCR was evaluated using an XF Cell Mito Stress Kit (Agilent). 5 × 10⁴ CD8 TILS isolated from control or Pant-treated murine tumors were analyzed using the T cell Metabolic Profiling kit on Seahorse HS Mini Xfp.i.

ECAR and OCR were measured using a Seahorse XF-24 Metabolic Flux Analyzer. Myofibroblasts cell lines were seeded at 2 × 10⁴ cells/well on Seahorse XF24 V7 multi-well plates for 16 h at 37°C in 10% CO₂. For the ECAR assay, 1 h before measurement, cell culture medium was replaced with DMEM (0 mM glucose) supplemented with 2 mM glutamine and incubated at 37°C in a non-CO₂ incubator. Cysteamine (500 μM) was added as indicated at this phase of treatment. ECAR was measured under basal conditions and after sequential addition of 10 mM glucose, 1 μM oligomycin, and 100 mM of 2-deoxyglucose. To record mitochondrial activity, the same assay medium was used supplemented with 1 mM sodium pyruvate and 10 mM glucose. OCR was measured before and after sequential injections of 1 μM oligomycin, 1 μM (carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone) FCCP, and 0.5 μM of antimycin A/rotenone. ECAR and OCR data were normalized to cell numbers using a cell titration curve quantified by crystal violet staining.

C2C12 cell mitochondrial respiration, basal respiratory capacity, coupling efficiency, proton leakage, and spare respiratory capacity were profiled by an XF24 metabolic flux analyzer (Seahorse Bioscience, Billerica, MA, USA). Furthermore, the oxygen consumption rate (OCR) was assessed in C2C12 cells that had been induced to undergo differentiation while being continuously treated with the ATP synthase inhibitor oligomycin (Wako Pure Chemicals), the uncoupling agent carbonyl cyanide p-(trifluoromethoxyl)-phenyl-hydrozone (FCCP; StressMarq Biosciences Inc., Victoria, BC, Canada), and a mitochondrial complex inhibitor rotenone (Sigma-Aldrich)/antimycin (Enzo Life Sciences, Plymouth Meeting, PA, USA). Fatty acid β-oxidation was evaluated by measuring the OCR when differentiated C2C12 cells were treated with bovine serum albumin–conjugated palmitate after 1 h of equilibration in Krebs-Henseleit buffer containing 0.5 mM carnitine (Tokyo Chemical Industry, Tokyo, Japan) and 2.5 mM glucose.