Activation of Mouse Bone Marrow-Derived Macrophages in Response to Antibodies

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Bone Marrow Macrophage Derivation and Activation with Antibodies

1. Perform euthanasia using CO₂ asphyxiation.
   1. Place the mouse in the induction chamber. Euthanize the mouse with 5% isoflurane anesthesia, for 60 – 90 s, until immobile and breathing is deep and slow.
   2. Turn off isoflurane anesthesia and administer 6 – 8 L/min of CO₂ until the mouse has stopped breathing. Leave the mouse with CO₂ on for at least another 5 min. Verify that there is no longer a heartbeat or respiration. Perform a cervical dislocation to ensure death.
      NOTE: 8 – 12 weeks-old mice provide the highest yield of macrophages.
2. Spray mouse legs with 70% ethanol and pin them with arms and legs stretched out in the supine position over a foam board. With scissors and forceps to hold the skin, make a shallow cut (0.2 cm) to remove the skin and fur from the surface of each of the hind legs.
   NOTE: Perform this procedure and all tissue culture manipulations in a sterile hood.
3. Trim muscle to make tibias and femurs visible. Remove the tibias and femurs, by cutting the bone and muscles just below the hip joint and above the ankles. Trim as much muscle off as possible.
4. Spray bones with 70% ethanol and wait 1 min for it to evaporate, then place them in a 6-well plate of bone flush medium (Iscove's modified Dulbecco's medium (IMDM), 10% fetal bovine serum (FBS)).
5. Trim the ends of the bones by cutting 0.1 cm of bone off the ankle and top of the femur so that the bone cavity is exposed. Ensure that the marrow is visible and a 26-gauge needle can be placed in the bone cavity.
6. Cut the bones and muscles to separate the tibias and femurs from the knee joints. Insert a 26-gauge needle attached to a 10 mL syringe into the cavity. Flush the bone marrow into a 50 mL conical tube with 5 mL of bone flush medium. Flush two tibias and two femurs, from one mouse, into each 50 mL tube.
7. Pipette up and down several times or vortex the marrow at a slow speed to gently disperse clumps. Strings of marrow should be broken up into small (less than 0.5 mm) flecks of marrow. Fill the conical tube to 50 mL with the bone flush medium. Pipette contents of the conical tube into a 75 cm² tissue culture-treated flask, and incubate at 37 °C, 5% CO₂ for 1 h.
   NOTE: Mature macrophages and mesenchymal cells will become adherent to the flask and be discarded, while the desired hematopoietic progenitors remain in suspension.
8. Pipette the medium with hematopoietic progenitors into a 50 mL conical tube. Centrifuge the tube at 300 x g at room temperature for 5 min. Discard the supernatant and resuspend the pellet in 5 mL of macrophage colony-stimulating factor (MCSF) culture medium (IMDM, 10% FBS, 5 ng/mL MCSF, 100 U/mL penicillin/streptomycin, and 150 μM monothioglycerol (MTG)).
9. Count cells using a hemocytometer. Add medium to resuspend cells to a concentration of 0.5 x 10⁶ cells/mL, 1.5 x 10⁷ cells/flask in 30 mL of medium and pipette up and down gently. Pipette 30 mL of suspension into a new 75 cm² tissue culture-treated flask.
10. On day 4 and day 7 after initial culture in step 1.9, verify that the cells are adherent and slightly branched using an inverted phase contrast bright field microscope. Discard the medium containing the non-adherent cells. Wash the cells with IMDM one time and add 30 mL of MCSF culture medium.
11. When the cells are mature by day 10 (>95% positive for F4/80 and Mac-1), verify again that the cells are adherent and slightly.
12. To plate cells for stimulation, remove the culture medium from the flask and add 8 mL of enzyme-free, EDTA-based cell dissociation buffer (Table of Materials). Incubate the cells for 5 min at 37 °C, 5% CO₂.
13. Scrape cells gently, with a small amount of pressure, using a sterile cell scraper. Check in the microscope that cells have detached from the flask surface. Pipette dissociated cells into a 50 mL conical tube. Rinse the flask 3 times with 5 mL of IMDM and pool the rinse solution with the cells that were harvested. Centrifuge cells at 300 x g at room temperature for 5 min.
14. Resuspend the cell pellet in 3 mL of MCSF culture medium per 50 mL conical tube. Count viable cells using a hemocytometer. Resuspend cells at a concentration of 1 x 10⁶ cells/mL and plate 100 μL of cell suspension (1 x 10⁵ cells/well) per well of a 96-well tissue culture treated, flat bottom plate.
    NOTE: Bones from one mouse will generate enough cells for 100 wells. If desired, 1 mL of cells can be plated in a 6-well plate (tissue culture treated, flat bottom). A larger number of cells (1 x 10⁶ cells) may be useful for western blot analyses.
15. Once adherent, stimulate duplicate or triplicate wells each with 10 ng/mL of LPS in IMDM, 30 mg/mL of IVIg, or IVIg + LPS. Doses of LPS or IVIg can be titrated to optimize responses. Leave duplicate or triplicate wells as unstimulated controls. Incubate them for 24 h (37 °C, 5% CO₂).
    NOTE: Re-plated cells should adhere to tissue culture wells within 1 h.
16. Collect cell supernatants from each well into individual 1.7 mL microcentrifuge tubes and remove any particulate matter by spinning at 10,000 x g for 5 min.
17. Remove the cell supernatant and avoid disturbing the pellet. Place the clarified cell supernatant in a sterile microcentrifuge tube.
    NOTE: Cell supernatants can be assayed for cytokines, IL-10, and cytokine subunit, IL-12/23p40, by enzyme-linked immunosorbent assay (ELISA) immediately or stored at -80 °C long term. Follow the ELISA protocol from a commercially available kit (Table of Materials). Other pro-inflammatory cytokines can be assayed, such as IL-6 and TNF, as they are also reduced by co-treatment with IVIg + LPS stimulation compared to stimulation with LPS alone. After stimulation, adherent cells can be prepared for techniques such as western blotting or quantitative polymerase chain reaction (Q-PCR).