Overview
This video demonstrates a method to study the responses to Mycobacterium tuberculosis infection in human M1 and M2 polarized monocyte-derived macrophages. The use of flow cytometry enables the study of a variety of surface markers associated with infected and uninfected macrophage populations.
Protocol
1. Mtb infection of monocyte-derived cells
NOTE: The following steps must be performed in a BSL-3 facility.
- Resuspend the Mycobacterium tuberculosis (Mtb) pellet in serum-free RPMI medium in a new sterile 50 mL tube and adjust the final bacterial concentration to approximately 5 x 106 CFU/mL.
- Remove the cell culture medium from the 6-well plate(s) containing monocyte-derived cells. Add 1 mL of serum-free RPMI medium to each well. Add 1 mL of bacterial suspension per well to obtain a multiplicity of infection (MOI) 5:1, i.e., 5 x 106 CFU per 106 macrophages in 2 mL/well and incubate the plates for 4 h at 37°C and 5% CO2.
- After infection, wash the cells 3 times with 1 mL of sterile wash buffer to remove extracellular bacteria. Tilt the plate and carefully remove the entire wash buffer from the corners. Resuspend the Mtb-infected monocyte-derived cells in 2 mL of RPMI complete medium without antibiotics and proceed to flow cytometry staining or incubate the cells for another 24 h (or other time-points) before flow cytometry.
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Materials
| Name | Company | Catalog Number | Comments |
| 8-well chamber slides | Lab-Tek | 154534 | |
| RPMI 1640 | Life Technologies Corporation | SH30096.01 | |
| IFN-γ | Peprotech | 300-02 | |
| Na-pyruvate | GE Healthcare Life Sciences | SH300239.01 | |
| L-Glutamine | GE Healthcare Life Sciences | SH30034.01 | |
| Falcon 6-well Flat Bottom plates | Corning Life Sciences | 353046 | |
| Fetal bovine serum (FBS) | Sigma-Aldrich | F7524 | |
| GM-CSF | Peprotech | 300-03 |
