Flow Cytometry Staining of Mycobacterium tuberculosis Infected Macrophage Subpopulations

Abstract

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 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.

Protocol

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.

1. 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 °C and 5% CO₂.
2. Gently pipette up and down a few times to ensure that the cells are detached. If possible, confirm cell detachment with microscopy. Transfer ....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Formaldehyde	Sigma-Aldrich	F8775
Goat anti-mouse IgG Alexa Fluor 594 secondary antibody	Invitrogen	R37121	Secondary antibody for CD64
Goat anti-Rabbit IgG Alexa Fluor 594 secondary antibody		A-11037	Secondary antibody for CD163
Fetal bovine serum (FBS)	Sigma-Aldrich	F7524
EDTA (0.5 M)	Karolinska University hospital, Huddinge	N/A
BD Comp bead plus	BD	560497