Macrophages play a pivotal role in the innate immune response of swine against various pathogens. Isolating and identifying porcine macrophages is crucial for understanding their functions in immunity and inflammation. In this protocol, we provide detailed methods for the isolation and identification of porcine macrophages, along with important notes to ensure successful experimentation.

Creative Biolabs outlines the procedures for isolating and identifying porcine macrophages, enabling researchers to investigate their functions in different contexts.

Materials

• Any suitable rabbit strains
• Sterile phosphate-buffered saline (PBS)
• Dulbecco's modified eagle medium (DMEM)
• Fetal bovine serum (FBS)
• Penicillin-streptomycin solution
• L-Glutamine
• Histopaque-1077
• Hank's balanced salt solution (HBSS)
• Trypsin-EDTA solution
• Red blood cell lysis buffer
• RPMI-1640 medium
• Macrophage activation stimulants

Methods

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

1. Isolation of Porcine Peripheral Blood Mononuclear Cells (PBMCs)
   Collect whole blood from a healthy pig in a sterile syringe containing anticoagulant and transfer it to a sterile tube. Dilute the blood 1:1 with PBS and layer it carefully over Histopaque-1077 in a new tube. Centrifuge the tube and carefully collect the PBMC layer, which should be at the interface of Histopaque-1077 and plasma, and transfer it to a new tube. Wash the PBMCs and resuspend the PBMCs in RPMI-1640 medium.
2. Differentiation of PBMCs into Macrophages
   Plate PBMCs in tissue culture flasks or plates in RPMI-1640 medium supplemented with FBS. Incubate the cells for 2-3 hours to allow monocytes to adhere. Remove non-adherent cells by gently washing with warm PBS. Add fresh RPMI-1640 medium with FBS and appropriate macrophage activation stimulants to the adherent cells. Incubate the cells for 48-72 hours to allow differentiation into macrophages.
3. Identification of Porcine Macrophages
   Assess macrophage morphology using a microscope. Perform flow cytometry to confirm the phenotype of macrophages. Stain cells with antibodies against porcine macrophage surface markers such as CD14 and CD163. Assess macrophage function by measuring cytokine production, phagocytosis, and other relevant functional assays.

Notes

• Use blood from healthy pigs to obtain high-quality PBMCs.
• Adjust cell density and culture conditions based on the specific experimental requirements.
• Check cell viability using a hemocytometer or trypan blue exclusion before proceeding with experiments.
• Choose appropriate macrophage activation stimulants based on the research question.
• Perform rigorous quality control measures to ensure accurate identification and characterization of macrophages.

In conclusion, this protocol provides a step-by-step guide for the isolation and identification of porcine macrophages. Researchers can utilize these methods to study the functions and roles of porcine macrophages in various immune responses and disease models. Creative Biolabs gives researchers technical and professional support, and you can contact us to learn more.