In Vitro Polarization of Murine Macrophage Protocol

Macrophages are tissue-resident professional phagocytes, which play an important role in immune-regulation. Activated macrophages are routinely classified into two different types: M1-macrophages (classic activation) and M2- macrophages (alternative activation), which perform pro- and anti-inflammatory active respectively.

In vitro polarization of macrophages is achieved by stimulation in the presence of appropriate activation factors. Here we describe a comprehensive protocol to obtain specific macrophage subtypes through in vitro polarization from murine bone marrow derived and peritoneal macrophages.

Procedures of In Vitro Polarization of Murine Macrophage Protocol

Day 1: Macrophages isolation

1. Isolation of peritoneal macrophages

1) Euthanize the mouse (6 to 12-week old) by rapid cervical dislocation, and then clean the abdomen with 70 % ethanol.
2) Make an incision in the midline of the abdomen, and then expose the intact peritoneal wall.
3) Fill a 5-mL syringe with cold PBS (+ 3% FBS), and carefully inject into the peritoneal cavity.
4) Gently massage the peritoneum for several times to dislodge any attached cells into the PBS solution.
5) Slowly withdraw peritoneal fluid using the same syringe, and collet the fluid into a 50-mL tube on ice.

Skip to Step 3.

2. Isolation of bone marrow derived macrophages

1) Euthanize the mouse (6 to 12-week old) by rapid cervical dislocation, and then clean the hind legs with 70 % ethanol.
2) Harvest femur and tibia by cutting off the hind legs close to the hip joint and cutting off the feet along.
3) Soak femur and tibia with 70 % ethanol, and then wash with PBS.
4) Carefully cut both ends of each bone as the bone is easily to be broken.
5) Flush the bone with cold PBS (+ 3% FBS) by using 1-mL syringe.
Note: Each bone requires 10 mL PBS. Slowly press the end of syringe to minimize cell stress.
6) Filter with 70-μm cell strainer, and collet into a 50-mL tube on ice.

3. Centrifuge at 1,000 rpm for 10 min at 4 °C, and discard supernatant.
4. Prepare differentiation culture medium: 100 ng/mL M-CSF in DMEM+.
5. Resuspend the cells with 1 mL warm differentiation culture medium, and then adjust to 1×10⁶ cells/mL.
6. Plate 2 mL cell suspension in six culture plate.
7. Culture the cells at 37 °C and 5% CO₂ for 2 hours to allow adherence, and then gently remove the nonadherent cells by washing with warn PBS.
8. Culture the cells at 37 °C and 5% CO₂ for 6~7 days. Check for cell density and condition, and change for fresh DMEM+ during the culturing.

Day 7: Macrophage polarization

9. Prepare activation culture medium:
   
   • M1 activation culture medium: 50 ng/mL IFN-γ, 10 ng/mL of LPS in DMEM+.
   • M2a activation culture medium: 20 ng/mL IL-4 in DMEM+.
   • M2c activation culture medium: 20 ng/mL IL-10 in DMEM+.
10. Remove differentiation culture medium, and wash with warn PBS.
11. Add 2 mL activation culture medium, and culture the cells at 37 °C and 5% CO₂.
    
    • For RNA analysis, culture for 24 hours.
    • For protein analysis, culture for 28 hours.

Day 8~9: Macrophage analysis

12. Generally, macrophage subtypes can be analyzed via cytokines detection or biomarkers. The following approaches are alternative for Th subsets analysis:
    
    • Cytokine analysis by flow cytometry
    • Protein quantification by ELISA
    • Imaging analysis by IF
    • Gene expression analysis by real time PCR