Quantifying Mycobacterial Redox State

A total of 2 × 10⁵ adipocytes and preadipocytes were seeded in a 96-well black plate and were infected at an MOI of 1 for 24 h with a plasmid expressing pMrx1-roGFP2. Ten days postinfection, the infected cells were washed once in PBS, and a fluorescence reading for emission at 520 nm was taken by excitation at 390 nm and 490 nm. For oxidizing or reducing, the cells were treated with 10 mM cumene hydroperoxide (CHP) or 40 mM DTT for 15 min, and then a fluorescence reading was taken using a Tecan M200 Pro reader. A log-phase culture grown in 7H9 complete medium in vitro was taken as the control; it was washed once in PBS, and after resuspension of the pellet in PBS, 200 μl was transferred to the black plate in triplicate for fluorescence reading. Bacterial samples were either left untreated or treated with 10 mM CHP or 40 mM DTT for full oxidization or reduction in order to calculate the E_MSH value for this in vitro-grown culture or for Mtb^A and Mtb^P as described in reference 32 (link).