A Fully Differentiated Macrophage Infection Model of Monocyte-Derived Cells with Mycobacterium tuberculosis

Samenvatting

Source: Mily, A., et al. Polarization of M1 and M2 Human Monocyte-Derived Cells and Analysis with Flow Cytometry upon Mycobacterium tuberculosis Infection. J. Vis. Exp.(2020).

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.

1. 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.
2. 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.
3. 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.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
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