Xfe96 extracellular flux analyzer

The extracellular acidification rates (ECARs) of the HUVECs in different groups were analyzed using an XFe96 extracellular flux analyzer (Seahorse Bioscience, Billerica, MA, USA). The optimal seeding density of HUVECs ranges from 10,000 to 60,000 cells per well and gives confluency of approx. 95% overnight in a 96-well seahorse culture plate. ECAR was determined by adding glucose (10 mM), oligomycin A (1 μM), and 2-deoxy-d-glucose (1 M).

An XF^e96 Extracellular Flux Analyzer (Seahorse Bioscience, North Billerica, MA, USA) was used to analyze the metabolic potential of Aa-treated and untreated RPE cells. Briefly, cells were seeded in a Seahorse 96-well plate and allowed to attach and grow to confluence. The cells were then analyzed for the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). The instructions of the manufacturer were followed. Four measurements were performed before and five after a single injection with differing concentrations of Aa. After the Seahorse Assay, the cells were lysed in the assay plate and the complete protein amount in each well was measured using the bicinchoninic acid (BCA) assay (Pierce Biotechnology, Rockford, IL, USA) with bovina serum albumin as the standard. The BCA assay was performed according to the manufacturer's instructions, and the protein levels were used to normalize the Seahorse data for each individual well. The results were analyzed using the Seahorse software, and the change in OCR and ECAR before and after Aa injection was calculated to assess the cells' metabolic potential.

The oxygen consumption rate (OCR) and the extracellular acidification rate (ECAR) were measured by mitochondria stress test using an XFe96 extracellular flux analyzer (Seahorse Bioscience, North Billerica, MA, USA), as previously described [25 (link)]. Briefly, 5.0 × 10⁴ cells were seeded in XFe96-well plates overnight in respective complete media, and the OCR and ECAR were measured in Seahorse XF DMEM pH 7.4 (Agilent Technologies) that was supplemented with 10 mM D-glucose, 2 mM L-glutamine, and 1 mM sodium pyruvate. As indicated, the cells were treated with the following pharmacological modulators of mitochondrial respiratory chain proteins: 1.5 µM oligomycin, 2.0 µM carbonyl cyanide p-tri-fluoromethoxyphenylhydrazone (FCCP), 0.5 µM antimycin A, and 0.5 µM rotenone (Seahorse bioscience). A total of three basal rate measurements were taken (0–14 min) prior to injection of the compounds, followed by three measurements of OCR/ECAR following the injection of each drug. OCR and ECAR readings were normalized to total protein levels (BCA protein assay, Pierce) in each well. Each cell line was seeded in 96 wells per experiment and replicate experiments were carried out at least three times.

Oxygen consumption rates (OCRs) and extracellular acidification rates (ECARs) from in vitro generated Tc1 and Tc17 cells (0.5 × 10⁶ cells/well) were measured in non-buffered DMEM without phenol red (containing 25 mM glucose, 2 mM l-glutamine, and 1 mM sodium pyruvate) under basal conditions and in response to 1 µM oligomycin, 0.5 µM fluoro-carbonyl cyanide phenylhydrazone, and 1 µM rotenone + 1 µM antimycin A (Sigma). The cells were analyzed with the XFe-96 Extracellular Flux Analyzer (Seahorse Bioscience).