Yo pro 1 assay

ECAR and OCR were measured in real-time with Glycolysis Stress Test Kit and Mito Stress Test Kit, respectively, using the Seahorse XFe96 Analyser (Seahorse Biosciences, USA) according to the previous study [31 (link)]. Data were normalized by cell numbers that was measured by the YO-PRO^®-1 Assay (Thermo Fisher Scientific).

BV2 and B6M7 cells were seeded in XFe 96-well microplates (6000 cells/well) (Agilent Technologies, Sana Clara, USA) with/without 5 μM STF31 (C₂₃H₂₅N₃O₃S, Millipore, Watford, UK) for 24 h, followed by LPS + IFNγ (both 100 ng/ml) or IL-4 (20 ng/ml) stimulation for a further 24 h. Cells were washed and incubated in base medium (Agilent Technologies) at 37 °C for 1 h. Extracellular Acidification Rate (ECAR) and Oxygen Consumption Rate (OCR) were measured in real-time with Glycolysis Stress Test Kit and Mito Stress Test Kit respectively using the Seahorse XFe96 Analyser (Agilent Technologies) following manufacturer’s instructions. Data were normalized by cell numbers that was measured by the YO-PRO®-1 Assay (Thermo Fisher Scientific).

The principle of cell bioenergy testing is based on the continuous injection of substances with different properties into the cellular oxidative phosphorylation system to emphasize the metabolic activities of the living cells. The ECARs and OCRs were detected using a glycolysis stress test kit and a Mito stress test kit^{46 (link)}, respectively, on a Seahorse Extracellular Flux (XF-96) analyzer (Seahorse Bioscience, Billerica, MA, USA). Mitochondrial function was analyzed in the presence of classical modulators of the electron transport chain, including oligomycin, FCCP (carbonylcyanide p-trifluoromethoxyphenylhydrazone), and rotenone. The compounds used for glycolysis stress testing were glucose, oligomycin A, and 2-deoxy-d-glucose (2-DG; inhibits glycolysis). Normalized data for determining the number of cells were detected by YO-PRO®-1 Assay (Thermo Fisher Scientific). OCR was calculated in pmoles min⁻¹ and ECAR in mpH min⁻¹.