Metabolic Characterization of Polarized M1 and M2 Bone Marrow-derived Macrophages Using Real-time Extracellular Flux Analysis

1. Culturing and Polarizing Bone Marrow-derived Macrophages

1. Day 0: Isolate femur and tibia bones from 6-10 week old mice of interest (C57Bl/6 in this assay).
2. Remove muscle tissue from the bones and place the bones in a 9 cm petri-dish filled with ice-cold PBS.
3. Transfer the bones to a 9 cm Petri dish filled with 70% ethanol and gently shake the plate for 30 sec.
4. Transfer the bones to a fresh 9 cm Petri dish filled with ice-cold sterile PBS.
5. Cut the femur and tibia at both ends with sterile scissors.
6. Flush the bones with 10 ml ice-cold sterile PBS using a 10 ml syringe and a 25 G needle. Collect the bone marrow in a 50 ml tube.
7. Transfer the bone marrow cells to a new 50 ml tube using a 23 G needle.
8. Repeat this step with a 25 G needle. Note: These steps are needed to remove cell clumps and bone residues.
9. Centrifuge at 300 x g for 5 min at 4 °C.
10. Discard the supernatant and resuspend the cells in 40 ml cell culture medium (RPMI-1640 plus 2 mM L-glutamine, 10% FCS, penicillin (100 U/ml), streptomycin (100 µg/ml) and 15% filtered L-929 cell (ATCC,CCL-1)-conditioned medium containing M-CSF (or alternatively 10 ng/ml recombinant M-CSF instead of L-929 cell-conditioned medium).
    Note: The generation of L-929 cell-conditioned medium was detailed earlier in this journal in 2013.¹¹
11. Culture the cells from one mouse in two 15 cm Petri dishes (do not use cell culture-treated plates since macrophage will not detach from this plastic) in 20 ml culture medium per plate in a 37 °C, 5% CO₂ incubator.
12. Add 10 ml fresh cell culture medium on day 3, without removing the 20 ml older cell culture medium.
13. Replace the old medium of each plate with 20 ml fresh culture medium on day 6. Doing this, non-adherent cells will also be removed.
14. Day 8:
    1. Detach cells by incubating them 5 min at 37 °C in 10 ml citrate saline (135 mM potassium chloride plus 15 mM sodium citrate in H₂O, autoclaved) and wash with 10 ml PBS. Count mature bone marrow-derived macrophages (BMDM) on day 8 using a cell counter.
    2. Verify the formation of mature (CD11b+ F4/80+) BMDM by visual inspection or by flow cytometry. Block 10⁵ cells with 1/100 anti-CD16/CD32 in PBS for 20 min in 100 µl PBS, incubate with 1/200 anti-CD11b-FITC plus 1/200 anti-F4/80-APC-Cy7 at RT, wash and analyze by flow cytometry.
       Note: Culturing mouse bone marrow-derived macrophages has been published in detail earlier in this journal by Ying et al.¹¹.
15. Seed 50,000 cells (optimal cell counts need to be optimized) per well in a cell culture microplate in 100 µl culture medium. Do not seed cells in background correction wells (A1, A12, H1, H12) and put medium only (no cells) in these wells. Tip: Hold the pipette at an angle about halfway down the side of the wells for most homogeneous cell layer.
16. Allow the cells to settle at RT in a cell culture hood for 1 hr. This will promote even cell distribution and reduce edge effects. Monitor adherence using a microscope and culture in a 37 °C, 5% CO₂ incubator.
17. Three hr after plating, stimulate cells for 24 hr with 10 ng/ml LPS, or 20 ng/ml recombinant mouse IL-4 to generate M1 or M2 macrophages, respectively. Include untreated naïve (M0) macrophages as a control. Assess proper M1 and M2 macrophage polarization by measuring LPS-induced secretion of IL-6, IL-12, TNF (ELISA) and IL-4-induced arginase-1 activity and/or MMR (CD206) and MGL (CD301) surface expression (flow cytometry) as described earlier^3,11.
    Note: At this point, cells can be (pre)-treated with pharmacological compounds of interest or macrophages can be stimulated with other factors. Since, the variation between separate wells might be substantial, it is advisable to use at least 4 and ideally 6 wells per condition.

2. Preparation of the Extracellular Flux Assay

1. Hydrate the sensor cartridge one day before running the assay:
   1. Place the sensor cartridge upside down next to the utility plate.
   2. Fill each well of the utility plate with 200 µl of calibrant solution and lower the sensor cartridge onto the utility plate submerging the sensors in the calibrant solution.
   3. Verify the calibrant solution level is high enough to keep the sensors submerged and place in a non-CO₂ 37 °C incubator O/N.
2. Prepare assay medium on the day of the assay as follows:
   1. Warm 50 ml XF base medium to 37 °C.
   2. Add 500 µl 200 mM L-glutamine to get a 2 mM final concentration.
   3. Adjust pH to 7.4 using 1 N NaOH and sterilize with a 0.2 µM filter and keep the assay medium at 37 °C.
3. Gently remove the cell culture medium from the polarized macrophages and store it at -20 °C for future use (e.g. ELISA to examine proper macrophage polarization). Wash the cells with 100 µl assay medium, add 180 µl assay medium per well and verify under the microscope that cells were not washed away. Place the cell culture plate in a 37 °C incubator without CO₂ for 1 hr prior to the assay run.
4. Prepare 10 x injection compound mixtures A till D as described in Table 1, warm to 37 °C, adjust the pH to 7.4 and filter sterilize.
   1. Make all compounds in these mixtures at 10x the final concentration in the cell culture wells and optimize these concentrations for each cell type.
5. Load the sensor cartridge using the provided loading guides with indicated volumes (Table 1) of the prepared 10x injection compound mixtures in ports A, B, C and D, respectively.

Table 1. Injection mixtures.

3. Bioenergetic Characterization of Polarized M1 and M2 BMDM using a Extracellular Flux Analyzer

1. Create an assay template with the assay wizard with 2 minute MIX and 3 minute MEASURE times and (at least) 3 mix and rate measurements loops before each of the 4 injections (Table 1) and after the last injection.
   Note: These concentrations are valid for bone marrow-derived macrophages and Raw264.7 macrophage cell lines, but should be optimized for each cell type. Adding pyruvate in the FCCP mixture is needed to fuel maximal respiration.
2. Start the run by pushing the start bottom and follow the instructions of the apparatus. First load the cartridge plate with the hydrated probes to allow calibration by apparatus and next load the cell plate.
3. After the assay, carefully discard all assay medium and store the analyzed cell culture microplate at -20 °C for future cell normalization using the cell proliferation assay kit as described in detail by the supplier.
   1. Briefly, prepare a 1x cell-lysis buffer by diluting component B 20-fold in distilled water and dilute compound A 400-fold in the 1x cell-lysis buffer. Add 200 µl per well, incubate 5 min at RT and measure fluorescence with ~180 nm excitation and ~520 nm emission maxima.
      Note: When working with non-adherent cells or if poor adherence is a concern, the direct cell proliferation kit can be used as an alternative since this protocol does not require discarding all assay medium. Yet, this direct protocol is slightly less sensitive compared to the protocol used in this lab.
4. After fluorescence measurement, normalize the cell count in each well as a ratio in which the average cell count of all naïve macrophage wells is set at 1.
   Note: Protein quantification (e.g. using a BCA assay) could be used as an alternative way to normalize cells counts. Yet, in our hands this assay was less sensitive compared to the cell proliferation assay kit.