Polarization of M1 and M2 Human Monocyte-Derived Cells and Analysis with Flow Cytometry upon Mycobacterium tuberculosis Infection

Human peripheral blood from healthy anonymous blood donors was obtained from the blood bank at Karolinska University Hospital, Huddinge, Sweden (ethical approval Dnr 2010/603-31/4). All experimental steps involving live virulent Mtb were performed at the Biosafety Level-3 (BSL-3) laboratory at the Public Health Agency of Sweden (FOHM), Solna, Sweden.

1. Preparation of media, buffers, and bacterial cultures

NOTE: Details about all the reagents and consumables are provided in the Table of Materials.

1. RPMI complete medium: Supplement RPMI 1640 with 1 mM sodium pyruvate, 2 mM L-glutamine, 10 mM HEPES, and 10% heat-inactivated fetal bovine serum (FBS). Avoid antibiotics in the cell culture medium when working with Mtb infection.
2. Serum-free RPMI medium: Supplement RPMI 1640 with 1 mM sodium pyruvate, 2 mM L-glutamine and 10 mM HEPES.
3. Wash buffer: Prepare phosphate buffer saline (PBS) containing 0.05% (v/v) Tween-80.
4. FACS buffer: Prepare PBS containing 2.5% (v/v) FBS and 0.5 mM EDTA.
5. Fixation buffer: Prepare PBS containing 4% formaldehyde to PBS. Ensure it is freshly prepared before use, e.g., mixed from a stock solution of 37% formaldehyde.
6. Permeabilization buffer: Add 0.1% sodium citrate and 0.1% Triton X-100 to deionized water.
7. Wash buffer (for immunofluorescence): Prepare PBS containing 0.1% BSA and 0.1% Tween-20.
8. Blocking buffer: Prepare PBS containing 0.1% BSA and 10% normal goat serum (NGS) to PBS.
9. Staining buffer (for immunofluorescence): Prepare PBS containing 0.1% BSA to PBS.
10. TB complete medium: Supplement Middle Brook 7H9 broth with 0.05% (v/v) Tween-80, 0.5% (v/v) glycerol, kanamycin (20 µg/mL), 10% (v/v) Middlebrook oleic acid, albumin, dextrose and catalase enrichment (Middlebrook OADC Enrichment).
11. Bacterial cultures: Use the standard virulent Mtb laboratory strain, H37Rv, constitutively expressing green fluorescent protein (GFP), for infection of monocyte-derived cells. This Mtb strain carries a pFPV2 plasmid that contains a gene encoding GFP, as well as a gene for kanamycin resistance. The antibiotic resistance enables continuous selection of plasmid-expressing bacteria in cultures containing kanamycin. Store bacteria in TB complete medium and 70% glycerol (1:1 dilution) at -80 °C.

2. Peripheral blood mononuclear cell isolation from buffy coats

NOTE: Perform all work with human blood (potentially contagious) inside a class II biosafety cabinet. Inactivate residual blood products with disinfectants for 15 min before discarding. Blood was obtained from healthy volunteers in this case. This in vitro macrophage differentiation protocol was set up to include 10 x 10⁶ isolated PBMCs/donor/well. From each donor, one buffy coat contains about 50 mL of a concentrated leukocyte suspension originating from whole blood, which normally provides 500–800 x 10⁶ PBMCs from which approximately 10% or 50–80 x 10⁶ monocytes can be retrieved.

1. Load 15 mL of buffy coat blood on top of 15 mL of density gradient medium prepared in 50 mL tube. Slowly overlay blood on top of the density gradient layer by leaning the pipette tip to the wall of the tube.
2. Spin the tubes at 600 x g for 25 min at room temp (RT) with 0 acceleration and 0 deceleration.
   NOTE: Close lids carefully before centrifugation and always check the tube holders for potential spill over after centrifugation.
3. Remove the top plasma layer with a sterile Pasteur pipette and thereafter carefully collect the mononuclear cell layer into a new 50 mL tube using a sterile Pasteur pipette. Add serum-free RPMI medium to the PBMC pellet to obtain a final volume of 50 mL. Mix carefully by inverting the tube a few times before centrifugation at 500 x g for 5 min at RT.
4. Discard the supernatant carefully and resuspend the cell pellet by flipping the bottom of the tube within the fingers.
5. To remove the density gradient medium contamination from the PBMCs, wash cells 2–3 times with serum-free RPMI to obtain a final volume of 50 mL. Centrifuge at 500 x g for 5 min at RT. Wash until the cell supernatant becomes transparent.
6. Discard the supernatant and resuspend the cells in 20 mL of serum-free RPMI medium.
7. Count the cells by trypan blue staining, manually using a hemocytometer or using an automated cell counter. Dilute the cell suspension in trypan blue in 1:2 or 1:10 dilution by mixing the cell-trypan blue sample in a 96 well plate e.g., 50 µL + 50 µL (for hemocytometer counting) or 10 µL + 10 µL (for automated cell counting) and count the cells to obtain the number of live cells/mL.
   CAUTION: Trypan blue is toxic and must be discarded in a separate chemical waste.

3. Differentiation and polarization of monocyte-derived cells

NOTE: For differentiation and polarization of monocyte-derived cells, a protocol that we previously established for M0, M1-like and M2-like cells as well as fully M1 and M2 polarized cells was followed²⁵. For simplicity, only fully polarized M1 and M2 macrophages are described here.

1. Use plastic adherence for isolation of monocytes. Briefly, seed freshly isolated PBMCs in a 6-well culture plate at an appropriate concentration, e.g., 10 x 10⁶ PBMCs/well in 2 mL serum-free RPMI medium and incubate at 37 °C and 5% CO₂.
2. After 2–3 h, remove the non-adherent cells with a pipette and wash the wells 3 times with 1 mL serum-free medium. The attached cells are monocytes and comprises around 10% of the total PBMCs added to the well, i.e., 10⁶ monocytes retrieved from 10 x 10⁶ PBMCs added per well.
3. For macrophage differentiation, prepare a working solution containing 50 ng/mL GM-CSF or M-CSF for M1 and M2 macrophage polarization respectively, added in 2 mL of RPMI complete medium per well. Culture the cells in a 5% CO₂ incubator in 37 °C for 3 days.
4. On the day 3, remove 1 mL of the cell culture medium carefully from the top layer of each well and supplement the cell cultures with 1 mL of fresh RPMI complete medium containing the double concentration of M-CSF or GM-CSF to obtain 50 ng/mL final concentration in the wells. Add the growth factors in a pre-made working solution of 100 ng/mL/well.
5. On the day 6, add different stimuli for the last 18–20 h of cell differentiation to obtain fully polarized and mature M1 (interferon-γ; IFN-γ, and lipopolysaccharide; LPS (E. coli O55:B5)) or M2 (interleukin 4; IL-4) macrophages. For M1 polarization, prepare IFN-γ and LPS in RPMI complete medium and add 50 µL per well to obtain a final concentration of 50 ng/mL IFN-γ and 10 ng/mL LPS in the cell cultures. For M2 polarization, prepare IL-4 in RPMI complete medium and add 50 µL per well to obtain a final concentration of 20 ng/mL in the cell cultures.
6. For differentiation of M0 polarized macrophages, stimulate the cells with M-CSF only, without any additional cytokines (providing an M2-like phenotype)²⁵.
7. Check the morphology of monocyte-derived cell cultures regularly with light microscopy to ensure that smaller monocytes are differentiated into larger macrophage-like cells. Also, monitor potential morphological differences between the M1 and M2 polarization, i.e., elongated and stretched M1 cells compared to M2 cells with a more rounded shape²⁵.
8. On the day 7, transfer the plates with monocyte-derived cells to a BSL-3 laboratory for infection with virulent Mtb.

4. Preparation of Mtb cultures

NOTE: The following steps must be performed in a BSL-3 facility. For all work with virulent Mtb, use protective clothing, respiratory protection, and ethanol resistant gloves.

1. Thaw a vial with 1 mL of bacterial aliquot and mix with 9 mL of TB complete medium (1:10 dilution) in a 50 mL filtered cap tube. Culture the suspension in an incubator at 37 °C and 5% CO₂.
2. After 24 h, spin the bacterial suspension at 2,300 x g for 10 min and carefully pour off the medium. Resuspend the bacterial pellet with 15–20 mL of fresh TB complete medium in a new 50 mL filtered cap culture tube and incubate at 37 °C and 5% CO₂. Mix the settled-down bacteria in the tube every 2–3 days to maintain a homogenous nutrient supply for all bacterial cells.
3. After 7–10 days, mix the bacterial suspension properly by pipetting up and down before transferring to a 50 mL screw cap tube.
4. Add 35–40 mL of sterile wash buffer to the 50 mL tube and spin the bacterial suspension at 2,300 x g for 10 min. Repeat the washing steps once. Resuspend the bacterial pellet in 1 mL of serum-free RPMI medium by pipetting with a micropipette.
5. Add another 9 mL of serum-free RPMI medium and sonicate the bacterial suspension inside a class II biosafety cabinet for 5 min at 37 °C, to disrupt the bacterial clumps. Dip the tube repeatedly (3–4 times) in the water bath sonicator to ensure maximum disruption of bacterial clumps. Measure the optical density (OD) of 1 mL of bacterial suspension at 600 nm wavelength using a spectrophotometer placed inside the biosafety cabinet. Use serum-free RPMI medium to set the reference.
6. Calculate the number of colony forming units (CFU) using the formula: (OD+0.155)/0.161 = Y, and Y x 10⁷= Y x 10⁶ CFU/mL, e.g., an OD value 0.32 provides a bacterial concentration of (0.32 + 0.155)/0.161 = 2.95, 2.95 x 10⁷= 29.5 x 10⁶ CFU/mL.

5. Mtb infection of monocyte-derived cells

NOTE: The following steps must be performed in a BSL-3 facility.

1. Resuspend the bacterial pellet in serum-free RPMI medium in a new sterile 50 mL tube and adjust the final bacterial concentration to approximately 5 x 10⁶ CFU/mL.
2. Remove the cell culture medium from the 6 well plate(s) containing monocyte-derived cells. Add 1 mL of serum-free RPMI medium to each well. Add 1 mL of bacterial suspension per well to obtain a multiplicity of infection (MOI) 5:1, i.e., 5 x 10⁶ CFU per 10⁶ macrophages in 2 mL/well and incubate the plates for 4 h in 37 °C and 5% CO₂.
3. After infection, wash the cells 3 times with 1 mL of sterile wash buffer to remove extracellular bacteria. Tilt the plate and carefully remove the entire wash buffer from the corners. Resuspend the Mtb-infected monocyte-derived cells in 2 mL of RPMI complete medium without antibiotics and proceed to flow cytometry staining or incubate the cells for another 24 h (or other time-points) before flow cytometry.

6. 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 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 in +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.

7. Flow cytometric data acquisition and analysis of Mtb-infected monocyte-derived cells

NOTE: Steps 7.1–7.2 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 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).

8. Immunofluorescence staining of Mtb-infected monocyte-derived cells

NOTE: Mtb infection must be performed in a BSL-3 facility.

1. For immunostaining, seed 2 x 10⁵ PBMCs/well in 500 μL of serum-free RPMI medium into 8 well chamber slides to obtain 2 x 10⁴ monocytes/well. After differentiation and M1/M2 polarization of monocytes, proceed with Mtb infection as described above. Fix the slides after 24 h of Mtb infection with fixation buffer for 30 min. Fixed slides are stored in the freezer at -20 ˚C until further analyses.
2. Wash the monocyte-derived cells twice with 200 µL of PBS for 10 min each.
3. Permeabilize the cells with 200 µL of permeabilization buffer for 5 min at RT.
4. Wash the cells 3 times with 200 µL of PBS for 5 min each.
5. Wash the cells twice with 200 µL of wash buffer for 5 min each.
6. Block non-specific binding with 200 µL of blocking buffer for 30 min at RT.
7. Dilute the primary antibodies 1:100 in staining buffer and incubate the M1 cells with an unconjugated CD64 antibody (Clone: 10.1) and the M2 cells with an unconjugated CD163 antibody (polyclonal) for 2 h at RT.
8. Next, wash the cells 3 times with 200 µL of wash buffer for 10 min each.
9. Dilute the fluorescent labeled secondary antibodies 1:1,000 in staining buffer and incubate the M1 cells with an anti-mouse IgG-Alexa Fluor 594 and the M2 cells with an anti-rabbit IgG-Alexa Fluor 594 for 1 h at RT.
10. Wash the cells 3 times with 200 µL wash buffer for 10 min each.
11. Remove the chamber grid and add ~20 μL of DAPI mounting medium in each well and put a 1.5 mm coverslip onto each slide.
12. Seal the coverslip with a layer of nail polish.
13. Acquire images using a confocal microscope with lasers emitting at 486 nm for excitation of GFP (green channel), 402 nm for DAPI (blue) and 560 nm for secondary antibody (red) respectively.