Macrophages are essential immune cells that play a pivotal role in the host defense against pathogens and in tissue homeostasis. Studying macrophages in non-human primates, such as monkeys, can provide valuable insights into their function and behavior in a physiological context closely related to humans. In this protocol, Creative Biolabs outlines a comprehensive method for the isolation and identification of monkey macrophages.

Materials

• Rhesus monkeys (Macaca mulatta) or other appropriate non-human primates
• Sterile phosphate-buffered saline (PBS)
• RPMI 1640 medium
• Fetal bovine serum (FBS)
• Collagenase D
• Monoclonal antibodies against monkey macrophage markers

Methods

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

1. Isolation of Monkey Macrophages
   Aseptically collect the tissue of interest (e.g., spleen, liver, or lung) and transfer it to the laboratory. Cut the collected tissue into small pieces using sterile scissors and forceps. Transfer the tissue pieces into a tissue homogenizer containing RPMI 1640 medium. Homogenize the tissue to obtain a single-cell suspension.
2. Macrophage Enrichment
   Centrifuge the cell suspension to pellet the cells. Discard the supernatant and resuspend the cell pellet in a solution of collagenase D and DNase I in RPMI 1640 medium. Incubate the cell suspension for 30-60 minutes with periodic agitation to digest the tissue and release macrophages.
3. Percoll Gradient Centrifugation
   Prepare a Percoll gradient by layering different concentrations of Percoll in FACS buffer. Carefully overlay the digested cell suspension onto the Percoll gradient. Centrifuge to separate macrophages, which will typically accumulate at the interface between the 44% and 67% Percoll layers. Collect the macrophage-rich interface layer and transfer it to a new tube. Wash, centrifuge and resuspend the collected cells with FACS buffer.
4. Identification of Monkey Macrophages
   Prepare a single-cell suspension of the isolated macrophages in flow cytometry buffer. Incubate the cells with monoclonal antibodies against monkey macrophage markers, such as CD68 and CD163. Analyze the stained cells using a flow cytometer. Observe the stained cells under a microscope for morphological confirmation of macrophages.

Notes

• The choice of tissue depends on the research objectives. Spleen and lymph nodes are commonly used sources for monkey macrophage isolation.
• Optimize collagenase and DNase I concentrations based on tissue type and size. Adjust digestion time accordingly.
• Optimize the Percoll gradient centrifugation conditions (e.g., centrifugation speed and time) based on the tissue source and cell yield.
• Isolated monkey macrophages can be further characterized through functional assays such as phagocytosis, cytokine production, and antigen presentation.
• Maintain aseptic conditions throughout the procedure to minimize contamination. Monitor cell viability using Trypan Blue exclusion.

This comprehensive protocol outlines the materials, methods, and important notes to successfully isolate and identify monkey macrophages for various research applications. Researchers can adapt and optimize these techniques to suit specific experimental requirements

Creative Biolabs is committed to advancing research through high-quality protocols and services, and we hope this protocol aids researchers in their studies involving monkey macrophages.