2.7. Mitochondrial and Glycolytic Stress Tests

A Seahorse XFe analyzer (Agilent Technologies) was used to measure the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) based on the mitochondrial stress test (Agilent 103015-100) and glycolysis stress test (Agilent 103020-100), respectively. Huh7 cells were plated in XF96 cell culture microplates at 2 × 10⁴ cells per well 16 h prior to infection with DENV2 at an MOI of 10. At 24 or 48 hpi, the culture media were replaced with 180 µL of XF base medium (Agilent Technologies) supplemented with 1 mM pyruvate, 2 mM L-glutamine, and with 10 mM glucose for the mitochondrial stress test or without glucose for the glycolysis stress test. The cells were rested for one hour in a non-CO₂ incubator at 37 °C, then placed in the Seahorse for analysis. The mitochondrial stress test used sequential injections of oligomycin (1 µM), p-trifluoromethoxyphenylhydrazone (FCCP, 1.5 µM), and rotenone and antimycin A (0.5 µM each). The glycolysis stress test used sequential injections of glucose (10 mM), oligomycin (1 µM), and 2-deoxyglucose (50 mM). Measurements were collected at 5 minute intervals; three times before and after injections and six times after the last injection. After analysis, cell numbers were measured by addition of 22 µL 10 nM Calcein-AM to all wells and absorption read at 520 nm after excitation at 488 nm in a SpectraMax M2^e plate reader (Molecular Devices). Calcein readings were imported into Wave (Agilent) software to normalize OCR and ECAR readings.

Mitochondrial activity was determined as follows: (1) Basal respiration was calculated using the last rate measurement before injection of oligomycin minus the non-mitochondrial respiration rate (defined as the minimum rate measurement after rotenone/antimycin A injection), (2) ATP production was calculated as the last rate measurement before oligomycin injection minus the minimum rate measurement after oligomycin injection, (3) Maximum respiration was calculated using the maximum rate measurement after FCCP injection minus non-mitochondrial rate, (4) Spare capacity was the maximal respiration minus basal respiration, and (5) Proton leak was the minimum rate measurement after oligomycin injection minus non-mitochondrial respiration. Glycolytic function was determined as follows: (1) Glycolysis was the maximum rate measurement before oligomycin injection minus the last rate of measurement before glucose injection, (2) Glycolytic capacity was the maximum rate measurement after oligomycin injection minus the last rate of measurement before glucose injection, (3) Glycolytic reserve was the glycolytic capacity minus glycolysis and (4) Non-glycolytic acidification was the last rate measurement prior to glucose injection.