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.
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