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Flow Cytometry Staining of Mycobacterium tuberculosis Infected Macrophage Subpopulations
In This Article
Overview
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.
- Gently pipette up and down a few times to ensure that the cells are detached. If possible, confirm cell detachment with microscopy. Transfer ....
Disclosures
Materials
| 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 |
This article has been published
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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)
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