This article details a method for analyzing M1 and M2 macrophage subpopulations infected with Mycobacterium tuberculosis using flow cytometry. The protocol involves cell detachment, antibody staining, and fixation to ensure accurate detection of infected cells.
Begin with adherent M1 and M2 macrophage subpopulations, distinguished by their specific surface markers. These cells are infected with fluorescent-labeled Mycobacterium tuberculosis.
Add FACS buffer to the culture wells.
EDTA in the buffer enables cell detachment from the culture wells.
Transfer the cell suspension to microcentrifuge tubes.
Centrifuge the tubes. Discard the supernatant. Resuspend the cells in a fluorochrome-conjugated anti-macrophage antibody cocktail and viability dye, Zombie-UV.
The Zombie-UV fluorescence dye enters dead cells through damaged membranes and binds to cytoplasmic proteins, enabling clear differentiation from viable unstained cells.
Further, the fluorescent-conjugated anti-macrophage antibodies bind to target antigens on the macrophages, enabling differential detection of subpopulations.
Wash the cells to remove unbound antibodies. Centrifuge. Remove the supernatant.
Resuspend the cells in a fixative buffer to cross-link proteins, preserving cell morphology, and inactivating Mycobacteria for safe handling during analysis.
Finally, centrifuge and process the samples for flow cytometry.
Use an appropriate gating strategy to determine the percentage of Mycobacterium-infected 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.
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