Culture of Human Monocyte-Derived Macrophages

The culture of human monocyte-derived macrophages (MDMs) is a tool for studying macrophages. Monocytes are known to be influenced by certain environmental cues to produce macrophages. Here Creative Biolabs describes a method for culturing monocyte-derived macrophages from blood monocytes.

Materials

• Peripheral blood
• Sterile phosphate-buffered saline (PBS)
• Magnetic-activated cell sorting (MACS) buffer
• Complete medium
• Cytokines

Methods

1. PBMC Isolation
   First, add sterile PBS to dilute the blood. Centrifuge at room temperature and discard the top plasma layer and collect the middle PBMC layer. Add sterile PBS to the PBMC and centrifuge to clean the PBMC, which can be repeated several times. Finally resuspend the cell pellet and count.
2. Monocyte Isolation
   Resuspend PBMCs to be isolated in cold MACS buffer and isolate and purify monocytes by magnetic bead sorting.
3. Culture of Monocyte-Derived Macrophages
   Resuspend the monocytes in the pre-warmed complete medium. Wash once in the medium and resuspend the monocytes. inoculate the monocytes into well plates, add complete medium. And depending on the cell type add cytokines such as GM-CSF, M-CSF, etc. and culture. On day 3, replace the medium with fresh medium. 6 days later the cells can be harvested.
4. Macrophage Identification
   Observe the harvested macrophages by microscopy. This can then be followed by the identification of monocyte-derived macrophages by flow cytometry or cellular immunofluorescence.

Notes

• Leukocyte cones is ideal for cell isolations, in particular monocyte isolation.
• We recommend seeding monocytes in a format that will be used for downstream treatments/application of MDMs so that there is no need to harvest and re-plate cells after differentiation.
• M-CSF or GM-CSF was required for monocytes to survive and differentiate into macrophages. This might differ between different types of media and cell culture plastics used.

We describe our protocols in a simple and clear manner for our clients to follow. You can also get more services by contacting us directly.

Reference

1. Rousselet, G. Macrophages. Springer New York, 2018.