Bioluminescence assay kit cl 2

M. tuberculosis mc²6230 were harvested at an OD₆₀₀ of 0.8 and IMVs made using the method described by Yano et al with slight modifications (Yano et al, 2011). Briefly, bacteria were lysed by 5 cycles of bead beating (1 min bead beating at 15,800 rpm followed by 2‐min cooling on ice) in a Biospec Bead Beater (Bartlesville, USA). The samples were then centrifuged at 59,860 g for 210 min. After snap‐freezing in liquid nitrogen, aliquots were stored at −80°C until use. Bradford reagent was used to estimate protein content which was shown to be 5 mg/ml.
All IMV experiments were carried out at 37°C in the same buffer that the IMVs were prepared in (10 mM HEPES, 50 mM KCl, 5 mM MgCl₂, 10% glycerol, pH 7) unless otherwise specified. ATP synthesis was measured using luciferase/luciferin (Roche Bioluminescence Assay Kit CL II). According to the kit protocols, luciferase/luciferin mix was combined with equal portions of buffer, IMVs (final concentration of 35 µg/ml), 250 mM NADH, 50 mM ADP, and 5 mM phosphate. Luminescence was monitored on a BioTek Synergy H4 plate reader in a 384‐well plate. For the drug‐treated wells, the IMVs were pre‐incubated with drugs for 10 min. Negative control lacked NADH (electron donor). The rate of ATP synthesis was determined by calculating the rate of change in luminescence over 15–30 min following the addition of all reagents.