Introduction

Monocyte derived macrophages are obtained through the process of monocyte to macrophage differentiation. When monocytes are isolated from blood and cultured in media with serum, they adhere and differentiate into macrophages. For optimal differentiation, it is recommended to add M-CSF to the culture media. Additionally, including factors such as IL-4, IL-10, or TGF-β can significantly enhance the viability of the differentiated macrophages.

Understanding the difference between monocytes and macrophages is crucial. Monocytes are precursor cells circulating in the bloodstream, while macrophages are differentiated cells residing in tissues, playing vital roles in immune response and tissue homeostasis. This distinction between macrophages vs monocytes underscores their unique roles and functions within the immune system. While macrophages originate from monocytes, their functions and locations differ significantly, highlighting the importance of studying both cell types in immunological research. This protocol helps provide a reliable method to achieve effective macrophage differentiation, facilitating the study of these essential immune cells. The protocol below is for a T25 or T75 flask but can be scaled down for 100 mm culture dishes or plates.

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Materials

Procedure
  1. Using aseptic techniques under sterile conditions, isolate PBMC from whole blood (See supplemental protocol A below).
  1. Prepare complete RPMI 1640 medium by supplementing RPMI 1640 medium with fetal bovine serum to a final concentration of 10%, 2 mM L-glutamine (if using medium not currently supplemented with GlutaMAX). Bring medium to 37°C. Optional: Supplement media with 1% penicillin-streptomycin (5,000 units/mL).
  1. Resuspend cell concentration to 2 x 106 cells/mL in complete RPMI 1640 medium.
  1. Transfer resuspended cell solution to a cell culture dish.
  1. Incubate the culture dish for 24 hours in 5% CO2 incubator at 37°C to allow the monocytes to adhere to dish.
  1. In a sterile conical tube, prepare complete RPMI 1640 medium with M-CSF Recombinant Human Protein at a final concentration of 40–50 ng/mL. Optional: Add 20 ng/mL IL-4 Recombinant Human Protein.
  1. Replace media in culture dish with the prepared media containing M-CSF and IL-4 (if using).
  1. Incubate cells for 6 days in 5% CO2 incubator at 37°C. Within the 6 days, replenish media with new complete RPMI 1640 medium supplemented with 40–50 ng/mL of M-CSF Recombinant Human Protein (Optional: and 20 ng/mL IL-4 Recombinant Human Protein) every 3–4 days. Check under microscope for cell health and confluence.
  1. Cells are ready to harvest when cells exhibit more granules in the cytoplasm and are a bit elongated. In addition, cells should be more adherent to the culture plate. When cells are ready to harvest, discard old media, and rinse dish twice with 1X PBS, discarding PBS after each rinse.
  1. Add 10 mL 10 mM EDTA to each culture dish, and let sit for 10 minutes, or until cells dissociate from dish at room temperature.
  1. Collect cells in a 50 mL conical tube and centrifuge at 300–400 x g for 4–5 minutes at room temperature.
  1. Discard the supernatant and rinse cells with 1X PBS.
  1. Centrifuge cells at 300–400 x g for 4–5 minutes.
  1. Discard the supernatant, and resuspend cells in flow cytometry staining buffer or desired medium.

Protocol tip:

Culturing monocytes on Nunc UpCell Dishes can help preserve cell viability and surface proteins during cell harvest. Nunc dishes with UpCell Surface enables harvesting of cells by temperature reduction without the need for dissociation enzymes (e.g., trypsin, EDTA), thereby maintaining cellular membrane and surface receptors.

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Supplemental protocol A: isolation of PBMC from whole blood

Materials

Procedure

  1. Dilute blood sample at least 1:1 with PBS in a conical tube.
  1. Underlay the diluted sample with a volume of Ficoll-Paque® medium that is equal to the original sample volume.
  1. Centrifuge at 400 x g for 20 minutes at room temperature with the brake OFF.
  1. Harvest PBMC located at the interface of the PBS and Ficoll-Paque® medium layers into a fresh tube.
  1. Fill the tube with PBS to wash the cells.
  1. Centrifuge the cells at 300–400 x g for 4–5 minutes at 2–8°C. Discard supernatant.
  1. Resuspend the cell pellet in an appropriate volume of flow cytometry staining buffer or buffer of choice, and perform a cell count and viability analysis.
  1. Centrifuge cells as in step 6, and resuspend in appropriate volume of complete RPMI 1640 so that the final cell concentration is 2 x 106 cells/mL.

Note:

As cells will be cultured, perform all steps using aseptic techniques, and use buffers that do not contain azide.

Protocol tip:

Significantly improve the accuracy of assessing cell health and concentration from freshly harvested peripheral blood mononuclear cells with automated counters. See detailed protocol on how to count PBMC using the Countess II FL Automated Cell Counter.

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Differentiation materials
Isolation from whole blood
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