Fluorescein diacetate requires metabolic conversion to fluorescein in order to become fluorescent. Only metabolically active 8-hydroxyquinoline Copper (WSDTY) tuberculosis cells are able to perform this conversion. Therefore, the accumulation of fluorescence is a measure of the vital state of individual cells without the need to rely on cellular multiplication. We essentially followed the staining procedure described previously. Briefly, M. tuberculosis mc26230 cells were harvested from cultures by centrifugation, resuspended in HdBSM or RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) and M. tuberculosis mc26230-specific supplements to an OD600 of 0.04, and treated with 1 or 10 μM 8HQ for 48 h at 37°C. Where indicated, CuSO4 was added to a final concentration of 7.5 μM. Then, 25 μl was removed, transferred into a fresh 96-well plate, and mixed with 2.5 μl FDA solution to give a final FDA concentration of 500 ng/ml. After 1 h of incubation at 37°C, staining was stopped by adding 200 μl PBS with 0.02% tyloxapol to each well. Samples were analyzed on a Guava EasyCyte flow cytometer using the green fluorescent channel. The flow cytometer was controlled by CytoSoft (v5.3) software.
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