In Vivo Activation of Peritoneal Macrophages in Response to Antibodies in a Murine Model

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

1. Challenging Mice in Vivo with intravenous immunoglobulin and lipopolysaccharide (IVIg+LPS) and Harvesting Peritoneal Macrophages

1. Weigh each mouse. Calculate the volume of IVIg (100 mg/mL stock concentration) needed to provide a final dose of 2.5 g/kg body weight. Calculate the amount of LPS (100 μg/mL stock concentration in Iscove's modified Dulbecco's medium (IMDM)) needed to provide a final dose of 0.2 μg/g body weight.        
   NOTE: For example, for a 20 g mouse, 500 µL of IVIg stock solution and 40 μL of a 100 μg/mL solution of LPS is required.
2. Draw the required amount of IVIg and LPS into a sterile 1 mL syringe fitted with a sterile 26 gauge needle. For control mice that will not receive IVIg, replace an equivalent volume of IVIg with sterile phosphate-buffered saline (PBS) pH 7.4.
3. Scruff the mouse with one hand by grabbing its skin at the back of the neck with your thumb and index finger and holding its tail and hind legs with your remaining fingers. Tilt its body toward the ground.
4. Place the needle at a 30 – 40° angle relative to the mouse's abdomen, in the lower right quadrant, bevel up, and insert 1.5 cm. Inject the IVIg + LPS, or PBS + LPS, intraperitoneally.
5. After 1h, perform euthanasia using carbon dioxide (CO₂) asphyxiation.
   1. Place the mouse in an 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.
6. Pin the mouse to a foam board, with limbs spread out. Spray the parts with 70% ethanol.
   NOTE: Perform the procedure in a sterile hood.
7. Make a shallow cut in the skin (0.2 cm) with scissors along the midline of the mouse, to avoid cutting the peritoneal cavity. Pull the abdominal skin off the mouse's belly using forceps, so that the lining of the intact peritoneal wall is visible.
8. Fill a 5 mL syringe with 5 mL of sterile PBS (pH 7.4). Insert a 25 or 27-gauge needle at the top of the cavity from either the left or right side toward the center of the mouse with the bevel of the needle up.
   NOTE: Place the needle carefully in the mouse so that you do not inject PBS into the organs, but rather, into the peritoneal space.
9. Push the fluid into the peritoneal cavity. Pull the needle out of the cavity and massage the peritoneum for 10 s to dislodge any cells. Insert the needle at the top of the cavity and avoid organs. Collect and place the lavage fluid recovered into a 15 mL conical tube on ice.
   NOTE: For every 5 mL of fluid injected, approximately 3.75 mL will be recovered.
10. Repeat injection and recovery (steps 1.8 – 1.9) 3 more times (total injection volume of 20 mL), to recover 15 mL of lavage fluid in total. Collect lavage from each mouse into a separate 15 mL conical tube.
    NOTE: After the final injection, it may be easier to extract the fluid from the far left and right sides of the mouse, where fluid has accumulated. The organs will cause less interference with the needle at this position.
11. Spin cells down at 300 x g for 5 min at 4 °C. Remove 1 mL of recovered lavage fluid and use for interleukin or IL-10 and IL-12/23p40 analyses by enzyme-linked immunosorbent assay (ELISA) according to manufacturer's instructions or freeze at -80 °C for future analyses.
    NOTE: To ensure the accurate comparison between treatments for lavage fluid cytokine analysis, perform 5 mL flushes of the peritoneal cavity 4 times for a final volume of 15 mL. Not all fluid will be recovered from each flush.
12. Resuspend the cells in 500 μL of plating medium (IMDM, 10% fetal bovine serum, FBS, and 100 U/mL penicillin/streptomycin) per mouse flushed. Count viable macrophages.         
    NOTE: The macrophages will be slightly larger than the other peritoneal cells. The typical yield is 5 x 10⁵ - 1 x 10⁶ macrophages/mouse using a wild-type C57BL/6 mouse.
    Optional: If a large number of red blood cells are present in the cell pellet, perform a lysis step according to the manufacturer's instructions to remove them.
13. Resuspend cells in the plating medium at 1 x 10⁶ macrophages/mL and plate 100 μL per well in a 96-well flat bottom tissue culture treated plate.        
    NOTE: It may be necessary to pool cells from more than one mouse for an experiment. For example, if one mouse had 5 x 10⁵ macrophages, 500 μL of plating medium would be required.
14. Incubate cells for 1 h at 37 °C, 5% CO₂ to allow macrophages to become adherent. The adherent cells are the peritoneal macrophages. Remove cell supernatants and non-adherent cells. Rinse the wells twice with 200 μL IMDM (pre-warmed to 37 °C), wait for 10 s, and slowly tilt the plate. Replace the plating medium. Incubate the cells at 37 °C, 5% CO₂ for 30 min prior to stimulation.
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. Samples may be used immediately or frozen and stored at -80 °C for cytokine analysis by ELISA.
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 ELISA immediately, or stored at -80 °C long term. Follow the ELISA protocol from the commercially available kit (Table of Materials). Other pro-inflammatory cytokines can be assayed, such as IL-6 and tumor necrosis factor (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).