The isolation and identification of canine macrophages play a pivotal role in advancing our understanding of immune responses in dogs and their relevance to various diseases. This protocol presents a comprehensive guide for the isolation and identification of these vital immune cells.

Materials

• Canine tissue samples (e.g., spleen, lymph nodes)
• RPMI-1640 medium
• Sterile phosphate-buffered saline (PBS)
• Fetal bovine serum (FBS)
• Penicillin-streptomycin solution
• Collagenase
• Dispase
• DNase I
• Trypan blue solution
• FACS buffer
• Macrophage-specific antibodies

Methods

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

1. Tissue Harvesting
   Obtain fresh canine tissue samples and place them in RPMI-1640 medium. Clean and mince the tissue into fine pieces using sterile instruments. Prepare a digestion mixture in medium. Incubate minced tissue in the digestion mixture with gentle agitation. Filter the digested tissue through a cell strainer to obtain a single-cell suspension.
2. Cell Isolation
   Centrifuge the suspension to pellet cells. Discard the supernatant and resuspend the pellet in medium. Layer the suspension over a density gradient medium to separate macrophages from other cell types. Mix cell suspension with trypan blue solution and count viable cells using a hemocytometer. Adjust the cell concentration as needed.
3. Surface Staining
   Incubate isolated cells with fluorescently labeled antibodies against macrophage markers (e.g., CD14, CD68). Wash cells with FACS buffer to remove unbound antibodies. Analyze stained cells using a flow cytometer to identify and quantify macrophage populations based on marker expression.

Notes

• The choice of tissue impacts the macrophage yield and phenotype. Different tissues harbor varying macrophage populations with specialized functions. Tailoring the tissue source to the research question is paramount.
• Adjust digestion time and enzyme concentrations based on tissue type and quality. Avoid over-triturating tissue during digestion to prevent cell damage.
• Maintaining appropriate culturing conditions is vital to sustain macrophage viability and function. Regular media changes and contamination monitoring are obligatory.
• Accurate identification of macrophage subsets demands expertise in flow cytometry. Regular calibration and compensation are indispensable.
• Integrating flow cytometry and microscopy data enriches the characterization of canine macrophages. Morphological insights complement marker-based identification.

Researchers can follow this protocol precisely and adapt it to specific experimental requirements. Creative Biolabs employs a multistep approach that combines innovative techniques with tried-and-true methods to obtain a highly pure and active macrophage population. Please do not hesitate to contact us.