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.
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.
2. Flow cytometric data acquisition and analysis of Mtb-infected monocyte-derived cells
NOTE: Steps 2.1–2.2 should be performed in advance of the flow cytometry staining described above. To avoid problems with cell clumping and dissociation of tandem dyes after cell fixation, sample acquisition of both Mtb-infected and uninfected cells is performed within 4-10 h after primary antibody staining.
Table 1: List of antibodies used for flow cytometry.
Laser | Filter | Fluorochrome | Phenotype | Function | Clone | Catalog no. | Company |
639 | 670/30 | AF647 | TLR2 | Pathogen recognition receptor | TL2.1 | 309714 | BioLegend |
639 | 780/60 | APC-Cy7 | CD206 | Mannose receptor | 15-2 | 321120 | BioLegend |
405 | 610/20 | BV605 | CD163 | Scavenger receptor | GHI/61 | 333616 | BioLegend |
405 | 670/30 | BV650 | CD80 | Co-stimulatory molecule | 2D10 | 305227 | BioLegend |
405 | 710/50 | BV711 | CCR7 | Chemokine receptor | G043H7 | 353228 | BioLegend |
405 | 780/60 | BV785 | CD86 | Co-stimulatory molecule | IT2.2 | 305442 | BioLegend |
488 | 530/30 | GFP | Mtb | Intracellular bacteria | |||
561 | 586/15 | PE | CD200R | Inhibitory receptor | OX-108 | 329306 | BioLegend |
561 | 620/14 | PE/DAZZLE 594 | CD64 | Fc gamma receptor-I of IgG | 10.1 | 305032 | BioLegend |
561 | 661/20 | PE-Cy5 (PC5) | HLA-DR | MHC class II molecule | L243 | 307608 | BioLegend |
355 | 450/50 | BUV395 | Viability Dye | Live/dead cell marker | Zombie UV | 423108 | Invitrogen |
The authors have nothing to disclose.
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 |