Flow Cytometry Staining of Mycobacterium tuberculosis Infected Macrophage Subpopulations
Published: November 30, 2023
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. 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.</…
Divulgazioni
The authors have nothing to disclose.
Materials
| 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 |
Citazione di questo articolo
Flow Cytometry Staining of Mycobacterium tuberculosis Infected Macrophage Subpopulations. J. Vis. Exp. (Pending Publication), e21801, doi: (2023).