Flow Cytometry Staining of Mycobacterium tuberculosis Infected Macrophage Subpopulations

To stain the cells for flow cytometry, incubate the infected cells with 1 milliliter of FACS buffer supplemented with 0.5 millimolar EDTA per well for at least 30 minutes. At the end of the incubation, collect the detached cells with gentle pipetting, and transfer the cells into individual screw-capped microcentrifuge tubes for centrifugation.

Resuspend the cells in approximately 50 microliters of fluorochrome-conjugated anti-human antibody cocktail and viability dye of interest, and incubate the cells for 30 minutes at 4 degrees Celsius protected from light.

After washing, fix the cells with 200 microliters of freshly prepared fixation buffer for 30 minutes at room temperature protected from light. After the fixation and washing, resuspend the cells in 400 microliters of FACS buffer, and transfer the cells to 1-milliliter microcentrifuge tubes.

To analyze the cells, use compensation beads to compensate the fluorescent signal for each fluorochrome-conjugated antibody in the cocktail, and use unstained cells to set the gate for the negative cell population. Then, acquire a minimum of 50,000 cells per sample.