2.8. Extracellular Flux Analysis

The oxygen consumption rates (OCR) of Mtb strains were measured using a Seahorse XFe96 Extracellular Flux Analyzer (Agilent Technologies Inc., New York, NY, USA). Mtb bacilli were adhered to the bottom of a Cell-Tak-coated XF cell culture microplate at 2 × 10⁶ bacilli per well. Cell-Tak has no effect on Mtb basal respiration [34]. Assays were carried out in unbuffered 7H9 media (pH 7.35) with no carbon source. Mtb bacilli were grown in this unbuffered 7H9 media, containing only 0.01% Tyloxapol, for 24 h before being seeded into the XF cell culture microplate at the start of the experiment. In general, basal OCR was measured for ~25 min before automatic sequential injection of various compounds through the drug ports of the sensor cartridge. The duration of OCR measurements after compound addition and the concentrations used varied by experiment. OASS modulation of Mtb OCR in the presence of L-Cys was performed by the simultaneous addition of Cys, OASS, and substrate OAS (final concentration of 4 mM, 0.03 µg/mL, and 4 mM, respectively). Q203-based modulation of the OCR in Mtb and ΔcydAB cells was performed in the presence of the indicated Cys concentration followed by the addition of Q203 (300 × MIC₅₀; MIC₅₀ for Q203 is 3 nM) [34]. To chemically complement Δcds1 cells with H₂S, different concentrations of NaHS were added to cells after measuring the baseline OCR. All OCR data figures indicate the time of each addition as dotted lines. OCR data points are representative of the average OCR during 4 min of continuous measurement in the transient microchamber, with the error being calculated from the OCR measurements taken from at least three replicate wells by the Seahorse Wave Desktop 2.2 software (Agilent Technologies Inc., New York, NY, USA). The transient microchamber was automatically re-equilibrated between measurements through the up and down mixing of the probes of the XF96 sensor cartridge in the wells of the XF cell culture microplate.