Xf96 extracellular flux analyzer

Real-time changes in extracellular acidification rate (ECAR) of HPAECs were analyzed with an XF96 Extracellular Flux Analyzer (Seahorse Bioscience, North Billerica, MA, USA) as described previously (27 (link)). Briefly, HPAECs were seeded at 2.5 × 10⁴ per well on Seahorse XF96 polystyrene tissue culture plates (Seahorse Bioscience) and incubated at 37 °C overnight in VCBM. The next day, after 1 μg/ml LPS treatment for 4 hours, the medium was changed to XF base Medium (Seahorse Bioscience) supplemented with 2 mM glutamine, and then the plate was incubated for 1 h in a non-CO₂ incubator at 37 °C. The ECAR assay was performed on the XF96 Extracellular Flux Analyzer (Seahorse Bioscience), and the ECAR values were normalized using protein concentration. Inhibitors and activators were used in these tests at the following concentrations: glucose (10 mM), oligomycin (2 μM), 2-DG (50 mM).

Extracellular acidification rate (ECAR), oxygen consumption rate (OCR), and fatty acid-β oxidation OCR (FAO-OCR) were measured as we performed previously (11 (link)). Briefly, 10,000 cells were plated in XF96 cell culture microplates (Seahorse Biosciences, Cat# 101104–004) in the respective growth medium. After 24 hr, the growth medium was replaced with XF assay medium (Seahorse Biosciences, Cat# 102365–100) followed by incubation at 37°C for 1 hr in a CO₂-free incubator. The basal ECAR and the ECAR following the addition of glucose (10 mM), oligomycin (1 μM), and 2-deoxyglucose (50 mM) were measured using an XF96 extracellular flux analyzer (Seahorse Biosciences) by following the manufacturer’s protocol. The ECAR values were normalized to total cell counts in each well. The basal OCR and the OCR following the addition of oligomycin (1 μM), FCCP (0.5 μM), and rotenone/antimycin A (0.5 μM) were measured using an XF96 extracellular flux analyzer (Seahorse Biosciences) by following the manufacturer’s protocol. The ECAR values were normalized to total cell counts in each well. FAO-OCR was measured by the XF Cell Mito Stress Test (Cat# 103015–100) using the XF Palmitate-BSA FAO substrate (Cat# 102720–100) in the presence or absence of etomoxir, an inhibitor of CPT1A, according to the manufacturer’s instructions. The OCR values were normalized to total cell counts in each well.