Cytation 3 cell imaging multiple mode reader

The BacTiter‐Glo™ Microbial Cell Viability Assay (Promega, USA) was used for quantitation of ATP content of bacteria cultures. Bacteria density was adjusted to OD₆₀₀ 0.05. 100 μl of culture was aliquoted into each well on a 96‐well white plate, and drug compounds were subsequently added into each well to make up to corresponding concentrations. DMSO concentration was kept at 1% across all wells. The plates were incubated at 37°C for 15 h. Subsequently, BacTiter‐Glo reagent was added to each well and incubated further for 12 min. The luminescence of each plate was measured using BioTek Cytation 3 Cell Imaging Multiple‐mode reader. Data analysis was performed on GraphPad Prism 7 software.

MIC₅₀ were determined as previously described (Kalia et al, 2017). 200 μl of mycobacteria culture (OD₆₀₀ 0.005) was dispensed into each well on 96‐well flat bottom plates. 2 μl of drugs is dispensed into each corresponding well. The assay plates were then incubated for 5–6 days at 37°C. The effect of various treatment on bacteria growth was determined by measuring turbidity at 600 nm on a BioTek Cytation 3 Cell Imaging Multiple‐mode reader.

MitoXpress^® Xtra was purchased from Luxcel Biosciences Ltd. Prior to the experiment, the probe was reconstituted in 1 ml sterile water. Cultures were diluted to OD₆₀₀ 0.5, and 150 μl was aliquoted into each well on a 96‐well black‐wall, clear‐bottom plate. 1.5 μl of test compounds was added to corresponding wells, and 10 μL probe was added to each well. The wells were sealed off with two drops of high sensitivity oil and incubated at room temperature for 10 min before placing the plate in Cytation‐3 Cell Imaging Multiple‐mode reader (BioTek, USA). Dual‐read time‐resolved fluorescence (excitation: 380 nm, emission: 650 nm) with two integration windows (30‐μs delay‐30‐μs measurement time; 30‐μs delay‐70‐μs measurement time) was measured every 5 min for 6 to 8 h at 37°C. The data from dual‐read time‐resolved fluorescence were used to calculate the fluorescence lifetime using the following transformation according to the manufacturer’s manual. Data analysis was performed on GraphPad Prism 7 software.