Disc3 5

The collapse of M. smegmatis Δψ was evaluated with the fluorescent, membrane potential sensitive probe 3,3′-dipropylthiodicarbocyanine (DisC₃(5), Life Technologies), as previously described with minor modifications^{48 (link),49 (link)}. M. smegmatis culture was adjusted to an OD₆₀₀ of 0.3. Prior to the assay, dextrose and nigericin were added to a final concentration of 10 mM and 1 µM, respectively. One hundred µL of culture was distributed per well in a clear-bottom, black-wall 96-well plate (Corning, Corning, NY, USA), followed by addition of DisC₃ (5) at a final concentration of 5 μM. DisC₃(5) quenching due to bacterial uptake was monitored by fluorescence at 600_ex/680_em, 30 °C in a Biotek Synergy HT multi-mode plate reader. Once fluorescence was quenched, bacteria were treated with DMSO, different concentrations of 2-AI compounds (2B8 and RA13: 31.25–125 µM), 15 µM CCCP, 18 µM BDQ, or 80 µM TRZ. Plates were continuously monitored to test for fluorescence reversal when DisC₃(5) is released, an indicator of bacterial membrane potential (Δψ) depolarization.

The membrane potential of S. Typhimurium grown in LB broth and EG medium to OD₆₀₀ of 0.5 was measured with the fluorescent probe DiSC₃(5) (Molecular Probes, Eugene, OR). The pellet of 1 mL of cells grown to log phase in LB broth or EG medium was resuspended in 5 mM HEPES, pH 7.2, supplemented with 5 mM casamino acids or 5 mM glucose, respectively. Samples were treated in 1 ml aliquots with 750 µM spermine NONOate for 15 minutes at 37°C. DiSC₃(5) was added to a final concentration of 1 µM from a stock solution made in DMSO. DiSC₃(5) was allowed to equilibrate in the cells before fluorescence measurements were collected in a Synergy 2 microtiter plate reader (BioTek, Winooski, VT) using excitation and emission wavelengths of 590 and 680 nm, respectively.