Flow Cytometry Staining of Mycobacterium tuberculosis Infected Macrophage Subpopulations

1. Flow cytometry staining of Mtb-infected monocyte-derived cells

NOTE: The following steps must be performed in a BSL-3 facility. The flow cytometry staining could be performed in a 96-well plate instead of tubes.

1. Detach the Mycobacterium tuberculosis, Mtb-infected cells (and uninfected controls) from the wells in the 6 well plate(s) by incubation with 1 mL of FACS buffer per well for at least 30 min at 37 °C and 5% CO₂.
2. Gently pipette up and down a few times to ensure that the cells are detached. If possible, confirm cell detachment with microscopy. Transfer the cell suspension from each well to a screw-capped microcentrifuge tube and spin the tubes at 200 x g for 5 min. Discard the supernatant carefully by pipetting.
3. Wash the cell pellet in each tube twice with FACS buffer and spin the cells at 200 x g for 5 min.
4. Stain the cells (about 0.5 x 10⁶ to 1 x 10⁶ cells/tube) with approximately 50 µL cocktail of fluorochrome-conjugated anti-human antibodies including TLR2 (AF647), CD206 (APC-Cy7), CD163 (BV605), CD80 (BV650), CCR7 (BV711), CD86 (BV786), CD200R (PE), CD64 (PE-Dazzle 594), HLA-DR (PE-Cy5) (Table 1) in combination with viability dye Zombie-UV for 30 min at 4 °C (refrigerator) in the dark.
5. Wash the stained cells twice with 400 µL of FACS buffer and spin the cells at 200 x g for 5 min.
6. Fix the stained cells with 200 µL of fixation buffer (freshly prepared) for 30 min at RT in the dark to ensure complete inactivation of mycobacteria.
7. Wash the cells twice with 400 µL of FACS buffer and spin at 200 x g for 5 min to remove excess fix buffer.
8. Resuspend the fixed cells in 400 µL of FACS buffer and transfer the samples into new 1 mL microcentrifuge tubes before taking them out of the BSL-3 laboratory for flow cytometry in BSL-2. Store the stained cells at +4 ˚C until sample acquisition.
   NOTE: Spray the tubes with 70% ethanol before taking them out of the BSL-3 laboratory. Formaldehyde is toxic (carcinogenic) and must be handled in a class II biosafety cabinet. Discard formaldehyde waste in a separate chemical waste.

2. Flow cytometric data acquisition and analysis of Mtb-infected monocyte-derived cells

NOTE: Steps 2.1–2.2 should be performed in advance of the flow cytometry staining described above. To avoid problems with cell clumping and dissociation of tandem dyes after cell fixation, sample acquisition of both Mtb-infected and uninfected cells is performed within 4-10 h after primary antibody staining.

1. Before flow cytometry staining described above, compensate the fluorescent signal for each fluorochrome-conjugated antibody listed in the staining panel (Table 1) using compensation beads (both positive and negative).
2. Titrate the antibody dilution for staining of human macrophages to obtain the optimal signal for each fluorochrome.
3. Use unstained cells to determine the level of background fluorescence necessary to set a gate for the negative cell population allowing for the stained cells to be visualized (macrophages are highly auto-fluorescent).
4. Acquire a minimum of 50,000 cells/sample in the flow cytometer using the recommended software for data acquisition.
5. Export the acquisition files from the flow cytometer in flow cytometry standard (FCS) format 3.1.
6. Analyze the FCS files in flow cytometry analysis software.
7. Gate macrophages according to their forward- and side scatter (FSC and SSC) characteristics and exclude dead cells by live/dead cell gating using the Zombie-UV viability dye.
8. Visualize H37Rv-GFP infected macrophages in the FITC channel.
9. Identify the frequency of positively stained cells and geometric mean fluorescence intensity (MFI) for all markers (Table 1).

Table 1: List of antibodies used for flow cytometry.