4.6. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) measurements

OCR and ECAR were measured in a XFe96 extracellular flux analyzer (Seahorse Bioscience). Cortical neurons were seeded in Agilent Seahorse XF96 cell culture microplate coated poly-l-lysine/laminin and incubated in a CO₂ incubator at 37 °C for 12 days before infection with recombinant HSV. After two days, 1 h prior to the assay, growth medium was replaced with XF assay medium containing Seahorse XF DMEM Medium (pH 7.4) (Agilent 103575-100), 10 mM Seahorse XF Glucose (Agilent 103577-100), 1 mM Seahorse XF Pyruvate (Agilent 103578-100), and 1× GlutaMAX (Gibco 35050-061) and the place was placed in a 37 °C incubator without CO₂. Basal respiration was calculated by subtracting the average OCR obtained after the third and fourth injections (2 μM Antimycin A+ 2 μM Piericidin A, and 50 mM 2-deoxy-d-glucose (2-DG), respectively) from the baseline OCR. Maximal respiration capacity was calculated by subtracting the average OCR obtained after the third and fourth injections from OCR obtained after carbonyl cyanide m-chlorophenyl hydrazone (CCCP) (12 μM) injection. ATP-production coupled respiration was calculated by subtracting the OCR obtained after injecting 2 μM oligomycin A from the basal OCR. Basal ECAR was calculated by subtracting the average non-glycolytic ECAR obtained after 2-DG injection from the baseline ECAR. Finally, the glycolytic capacity was calculated by subtracting the average non-glycolytic ECAR from ECAR obtained after injecting 2 μM oligomycin A.