Macrophages play a pivotal role in the immune system, acting as the first line of defense against pathogens and contributing to tissue repair and homeostasis. Isolating and identifying these versatile immune cells is crucial for advancing our understanding of immunology.

In this protocol, Creative Biolabs presents a comprehensive guide to the isolation and identification of rabbit macrophages, leveraging the expertise of expert advice. The method incorporates innovative techniques to ensure the purity and viability of the isolated macrophage population.

Materials

• Any suitable rabbit strains
• Sterile phosphate-buffered saline (PBS)
• Dulbecco's modified eagle medium (DMEM)
• Fetal bovine serum (FBS)
• Antibiotics
• Lymphocyte separation medium
• Trypan blue solution
• Antibodies

Methods

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

1. Isolation of Rabbit Monocytes
   Prepare a suitable animal housing environment following ethical guidelines. Euthanize the rabbit and collect peritoneal lavage fluid using ice-cold PBS. Transfer fluid to a sterile tube. Centrifuge the tube at low speed to pellet cells. Carefully aspirate the supernatant, leaving behind the cell pellet. Resuspend the pellet in PBS and mix the cell suspension with lymphocyte separation medium to incubate. Centrifuge to collect the mononuclear cell layer at the interface. Wash cells with PBS, centrifuge, and resuspend in DMEM supplemented with FBS and antibiotics. Plate cells in cell culture flasks and incubate.
2. Macrophage Enrichment
   After 2-3 hours, remove non-adherent cells by washing flasks with PBS. Optionally, for enhanced monocyte enrichment, use a cocktail according to the manufacturer's instructions. Culture adherent cells in DMEM with FBS and antibiotics, allowing monocytes to differentiate into macrophages.
3. Identification and Characterization
   Gently detach macrophages using a cell scraper for downstream analyses. Determine cell concentration using a hemocytometer. Perform flow cytometry analysis to confirm cell identity. Use antibodies against rabbit macrophage-specific markers such as CD14 and CD68.

Notes

• Assess cell viability at every step to ensure the quality of isolated macrophages.
• The use of a cocktail is optional but can enhance monocyte enrichment.
• Rabbit macrophages can exhibit various phenotypes based on tissue origin and activation state. Further studies may be necessary to elucidate their specific functions.
• Ensure that all experimental procedures involving animals are approved by the appropriate ethics committee.
• Maintain strict aseptic conditions during the entire procedure to prevent contamination.

This comprehensive protocol outlines a step-by-step approach for the isolation and identification of rabbit macrophages. It is provided for informational purposes. Researchers can adapt and optimize the protocol based on their specific experimental needs and available resources. Creative Biolabs gives researchers technical and professional support, and you can contact us to learn more.