Macrophages stand out as versatile sentinels of the immune system, playing a crucial role in both innate and adaptive immunity. The ability to isolate and identify these cells with precision is vital for unraveling the complexities of immune responses. Mouse macrophages are a commonly used cell population for research.

Creative Biolabs outlines a comprehensive approach to isolating and identifying mouse macrophages in the protocol below, emphasizing experimental design and attention to detail.

Materials

• Laboratory animals: genetically modified mice, wild-type mice
• Sterile phosphate-buffered saline (PBS)
• Dulbecco's modified eagle medium (DMEM)
• Fetal bovine serum (FBS)
• Penicillin-streptomycin solution
• Collagenase D and dispase
• Trypan blue solution
• Antibodies: anti-F4/80 antibody, anti-CD11b antibody

Methods

Fig.1 Methods for isolation and identification of mouse macrophages. (Creative Biolabs Original)

1. Tissue Harvesting
   Sacrifice mice humanely, adhering to ethical guidelines. Harvest the desired tissue (e.g., spleen, peritoneal cavity, or lungs) and place it in a sterile petri dish containing cold PBS. Mince the tissue into small pieces using scissors and transfer to a sterile tube.
2. Macrophage Isolation
   Digest tissue with a mixture of digestive enzymes in solution at 37°C for 30-60 minutes with periodic agitation. Filter the digested tissue through a 70 μm cell strainer to obtain a single-cell suspension. Wash the cells with PBS and centrifuge to obtain a cell pellet. Resuspend the pellet in appropriate culture medium.
3. Macrophage Identification and Characterization
   Plate cells in tissue culture plates and incubate in a humidified incubator to allow adherence of macrophages. Remove non-adherent cells and retain adherent macrophages. Detach adherent macrophages and stain with antibodies against CD11b, F4/80, and CD68. Analyze stained cells using a flow cytometer to quantify macrophage populations and assess their surface marker expression.

Notes

• Maintain cell viability throughout the isolation procedure by working under sterile conditions and using proper aseptic techniques.
• The digestion time can vary depending on tissue type and size. Monitor the digestion process for optimal results.
• Perform isolations and analyses in triplicate to ensure reproducibility of results.
• Adjust gating strategies based on the staining patterns of F4/80 and CD11b to precisely identify macrophage subsets.

This protocol offers a robust and comprehensive approach, allowing researchers to delve into the intricacies of macrophage biology. By incorporating the nuances of tissue selection, cell isolation, and advanced techniques like flow cytometry, Creative Biolabs is committed to excellence in macrophage research. You can get more information by contacting us directly.

References

1. Zhang, Xia, Ricardo Goncalves, and David M. Mosser. "The isolation and characterization of murine macrophages." Current protocols in immunology 83.1 (2008): 14-1.
2. Rios, Francisco J., Rhian M. Touyz, and Augusto C. Montezano. "Isolation and differentiation of murine macrophages." Hypertension: Methods and Protocols (2017): 297-309.