Flow Cytometry Staining of Mycobacterium tuberculosis Infected Macrophage Subpopulations

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.

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 the cell suspension from each well to a screw-capped microcentrifuge tube and spin the tubes at 200 x g for 5 min. Discard the supernatant carefully by pipetting.
3. Wash the cell pellet in each tube twice with FACS buffer and spin the cells at 200 x g for 5 min.
4. Stain the cells (about 0.5 x 10⁶ to 1 x 10⁶ cells/tube) with approximately 50 µL cocktail of fluorochrome-conjugated anti-human antibodies including TLR2 (AF647), CD206 (APC-Cy7), CD163 (BV605), CD80 (BV650), CCR7 (BV711), CD86 (BV786), CD200R (PE), CD64 (PE-Dazzle 594), HLA-DR (PE-Cy5) (Table 1) in combination with viability dye Zombie-UV for 30 min at 4 °C (refrigerator) in the dark.
5. Wash the stained cells twice with 400 µL of FACS buffer and spin the cells at 200 x g for 5 min.
6. Fix the stained cells with 200 µL of fixation buffer (freshly prepared) for 30 min at RT in the dark to ensure complete inactivation of mycobacteria.
7. Wash the cells twice with 400 µL of FACS buffer and spin at 200 x g for 5 min to remove excess fix buffer.
8. Resuspend the fixed cells in 400 µL of FACS buffer and transfer the samples into new 1 mL microcentrifuge tubes before taking them out of the BSL-3 laboratory for flow cytometry in BSL-2. Store the stained cells at +4 ˚C until sample acquisition.
   NOTE: Spray the tubes with 70% ethanol before taking them out of the BSL-3 laboratory. Formaldehyde is toxic (carcinogenic) and must be handled in a class II biosafety cabinet. Discard formaldehyde waste in a separate chemical waste.

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.

1. Before flow cytometry staining described above, compensate the fluorescent signal for each fluorochrome-conjugated antibody listed in the staining panel (Table 1) using compensation beads (both positive and negative).
2. Titrate the antibody dilution for staining of human macrophages to obtain the optimal signal for each fluorochrome.
3. Use unstained cells to determine the level of background fluorescence necessary to set a gate for the negative cell population allowing for the stained cells to be visualized (macrophages are highly auto-fluorescent).
4. Acquire a minimum of 50,000 cells/sample in the flow cytometer using the recommended software for data acquisition.
5. Export the acquisition files from the flow cytometer in flow cytometry standard (FCS) format 3.1.
6. Analyze the FCS files in flow cytometry analysis software.
7. Gate macrophages according to their forward- and side scatter (FSC and SSC) characteristics and exclude dead cells by live/dead cell gating using the Zombie-UV viability dye.
8. Visualize H37Rv-GFP infected macrophages in the FITC channel.
9. Identify the frequency of positively stained cells and geometric mean fluorescence intensity (MFI) for all markers (Table 1).

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

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