eoe sub articles

EoE Sub Articles

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.
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	<li>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 &#176;C and 5% CO2.</...

flow cytometry staining of mycobacterium tuberculosis infected macrophage subpopulations

Source: Mily, A., et al. <a href="https://app.jove.com/v/61807">Polarization of M1 and M2 Human Monocyte-Derived Cells and Analysis with Flow Cytometry upon Mycobacterium tuberculosis Infection.</a> J. Vis. Exp. (2020)
This video showcases a flow cytometric gating strategy utilizing a multi-color panel to analyze cell viability and specific fluorescent-conjugated anti-human antibody markers. This approach makes it possible to determine the percentage of Mycobacterium tuberculosis-infected cell populations accurately.

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 ...

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.