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All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.
1. Isolation of primary human monocytes by immunomagnetic negative selection
<ol>
	<li>Collect 40 mL of fresh, human whole blood (from either an HIV+&#160;patient or healthy control) in four 10 mL ethylenediaminetetraacetic acid (EDTA) vacuum tubes (10 mL of blood per tube). Using sterile techniques under a biosafety cabinet, transfer all 40 mL of blood into one 50 mL conical propylene tube.</li>
	<li>Following the manufacturer&#39;s protocol for the selected human monocyte isolation kit (Table of Materials), add 2 mL of monocyte isolation cocktail, provided in the kit, to the tube of blood. Vortex magnetic beads, also provided in the kit, for 30 s, and add 2 mL to the tube of blood.
	<ol>
		<li>If less than 40 mL of blood is available, scale down the reagents added. To mix the solution, pipette up and down with a plastic 25 mL serological pipette and incubate for 5 min at room temperature (RT).</li>
	</ol>
	</li>
	<li>Separate the blood mixture equally into four 50 mL tubes and add 30 mL of sterile phosphate-buffered saline (PBS) containing 1 mM EDTA to each tube. Mix by pipetting up and down with a plastic 25 mL serological pipette.</li>
	<li>Place the tubes in magnet holders for 10 min to remove the antibody-conjugated magnetic beads. Use four magnet holders simultaneously, one for each tube, to allow consistent incubation and isolation times for each blood sample.</li>
	<li>Draw up the contents from the center of each tube, using a pipette, while they are still in the magnet holders. Be careful not to draw up red blood cells (no more than 10% of the 10 mL starting volume), and place the contents into one of four new 50 mL tubes.</li>
	<li>Add 500 &#181;L of vortexed magnetic beads to each 50 mL tube. Pipette up and down with a 25 mL pipette and incubate at RT for 5 min. Then, place the tubes into magnet holders for 5 min.</li>
	<li>Carefully transfer the contents from the center of each tube while still in magnet holders into one of four new 50 mL tubes. Directly place each new 50 mL tube in the magnet holders for 5 min.</li>
	<li>Carefully transfer contents from the center of each tube into one of four new 50 mL tubes. Spin all new 50 mL tubes at 300 x&#160;g&#160;for 5 min. Aspirate the supernatant and resuspend all four cell pellets in a total of 10 mL of sterile PBS.</li>
	<li>Count cells by trypan blue exclusion using a hemocytometer. 
	NOTE: 8-20 x 106&#160;cells are generally obtained from 40 mL of whole blood.</li>
</ol>
2. Culturing of primary human monocytes
<ol>
	<li>Using a 37 &#176;C water bath, warm serum-free RPMI 1640 media supplemented with 1% penicillin-streptomycin (pen/strep), and (while continuing to use sterile techniques under a biosafety cabinet) resuspend the isolated monocytes in this media at a concentration of 1 x 106&#160;cells/mL.</li>
	<li>Add 1 mL of resuspended cells to each well of a 6-well plate or into a 35 mm dish (the final number of cells should be 1 x 106&#160;cells/plate), and place in a 37 &#176;C incubator with 5% CO2. Wait for 0.5-1.0 h for cells to adhere.</li>
	<li>Using a 37 &#176;C water bath, warm heat-inactivated (HI) fetal bovine serum (FBS). Add 100 &#181;L (10% final concentration) of FBS to each plate. Add growth factors to the cells to promote macrophage differentiation.
	<ol>
		<li>Prime macrophages for an M1-like phenotype by adding 25 ng/mL of granulocyte-macrophage colony-stimulating factor (GM-CSF) to media. Prime macrophages for an M2-like phenotype by adding 50 ng/mL of macrophage colony-stimulating factor (M-CSF) to media. 
		NOTE: Both GM-CSF and M-CSF allow monocyte differentiation to a general macrophage phenotype (M0) while priming cells for M1 or M2, respectively.</li>
	</ol>
	</li>
</ol>

<table><tbody><tr><td>0.5M EDTA</td><td>Invitrogen</td><td>AM9260G</td><td></td></tr><tr><td>BD Vacutainer Plastic Blood Collection Tubes with K2EDTA</td><td>BD Biosciences</td><td>366643</td><td></td></tr><tr><td>Easy 50 EasySep Magnet</td><td>StemCell Technologies</td><td>18002</td><td></td></tr><tr><td>Easy Sep Direct Human Monocyte Isolation Kit</td><td>StemCell Technologies</td><td>19669</td><td></td></tr><tr><td>Fetal Bovin Serum Defined Heat Inactivated</td><td>Hyclone</td><td>SH30070.03HI</td><td></td></tr><tr><td>Nunc 35mm Cell Culture Dish</td><td>Thermo Scientific</td><td>150318</td><td></td></tr><tr><td>PBS</td><td>Gibco</td><td>20012027</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Gibco</td><td>15140122</td><td></td></tr><tr><td>Recombinant Human GM-CSF</td><td>R&amp;amp;D Systems</td><td>215-GM-050</td><td></td></tr><tr><td>Recombinant Human M-CSF</td><td>R&amp;amp;D Systems</td><td>216-MC-025</td><td></td></tr><tr><td>RPMI 1640 with L-Glutamine</td><td>Corning</td><td>10040CVMP</td><td></td></tr></tbody></table>

in vitro culture and differentiation of primary monocytes into macrophages

Source: Plaisance-Bonstaff, K., et al. <a href="https://app.jove.com/v/59967">Isolation, Transfection, and Culture of Primary Human Monocytes.</a> J. Vis. Exp. (2019)
This video demonstrates the priming of primary monocytes into macrophages. Primary human monocytes can be isolated and differentiated to an M1-like pro-inflammatory phenotype using the granulocyte-macrophage colony-stimulating factor (GM-CSF) or to an M2-like anti-inflammatory phenotype using the macrophage colony-stimulating factor (M-CSF).

In Vitro Culture and Differentiation of Primary Monocytes into Macrophages

Source: Plaisance-Bonstaff, K., et al. Isolation, Transfection, and Culture of Primary Human Monocytes. J. Vis. Exp. (2019)
This video demonstrates the ...

Monocytes are immune cells that differentiate into macrophages and antigen-presenting dendritic cells to protect against infection.
To culture monocytes and induce their differentiation in vitro, begin by taking a suspension of primary human monocytes in a suitable culture medium. Add the cell suspension into the wells of a culture plate. Incubate at physiological conditions to allow the cells to adhere.
Add differentiation-inducing cytokines &#8212; granulocyte-macrophage colony-stimulating factor, or GM-CSF, to one well and macrophage colony-stimulating factor, or M-CSF, to another. Incubate for an appropriate duration.
GM-CSF binds to its heterodimeric receptor on the cells and induces the differentiation of the monocytes. GM-CSF binding results in the activation of intracellular signaling pathways, leading to the expression of genes for priming into an M1 macrophage-like phenotype.
The M1-like cells &#8212; rounded cells with a large cytoplasmic volume &#8212; are pro-inflammatory and assist in the defense against infection.
In the other well, the M-CSF binds to its homodimeric receptor on the monocytes and induces the differentiation of the cells. M-CSF binding results in the activation of intracellular signaling pathways different from GM-CSF, leading to the expression of genes for priming into an M2 macrophage-like phenotype.
The M2-like cells &#8212; with an elongated shape &#8212; are anti-inflammatory and exhibit tissue remodeling and repair functions.
The differentiated macrophage cells are ready for further downstream assays.

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Research

JoVE Encyclopedia of Experiments

Immunology

Immune Systems and Components

Cell Differentiation Techniques

536 Views. Source: Plaisance-Bonstaff, K., et al. Isolation, Transfection, and Culture of Primary Human Monocytes. J. Vis. Exp. (2019) This video demonstrates the priming of primary monocytes into macrophages. Primary human monocytes can be isolated and differentiated to an M1-like pro-inflammatory phenotype using the granulocyte-macrophage colony-stimulating factor (GM-CSF) or to an M2-like anti-inflammatory phenotype using the macrophage colony-stimulating factor (M-CSF). 

Video: In Vitro Culture and Differentiation of Primary Monocytes into Macrophages - Concept

Production and Characterization of Human Macrophages from Pluripotent Stem Cells

Macrophages are present in most vertebrate tissues and comprise widely dispersed and heterogeneous cell populations with different functions. They are key players in health and disease, acting as phagocytes during immune defense and mediating trophic, maintenance, and repair functions. Although it has been possible to study some of the molecular processes involved in human macrophage function, it has proved difficult to apply genetic engineering techniques to primary human macrophages. This has significantly hampered our ability to interrogate the complex genetic pathways involved in macrophage biology and to generate models for specific disease states. An off-the-shelf source of human macrophages that is amenable to the vast arsenal of genetic manipulation techniques would, therefore, provide a valuable tool in this field. We present an optimized protocol that allows for the generation of macrophages from human induced pluripotent stem cells (iPSCs) in vitro. These iPSC-derived macrophages (iPSC-DMs) express human macrophage cell surface markers, including CD45, 25F9, CD163, and CD169, and our live-cell imaging functional assay demonstrates that they exhibit robust phagocytic activity. Cultured iPSC-DMs can be activated to different&#160;macrophage states that display altered gene expression and phagocytic activity by the addition of LPS and IFNg, IL4, or IL10. Thus, this system provides a platform to generate human macrophages carrying genetic alterations that model specific human disease and a source of cells for drug screening or cell therapy to treat these diseases.

This protocol describes the robust generation of macrophages from human induced pluripotent stem cells, and methods for their subsequent characterization. Cell surface marker expression, gene expression, and functional assays are used to assess the phenotype and function of these iPSC-derived macrophages.

Macrophages are present in most vertebrate tissues and comprise widely dispersed and heterogeneous cell populations with different functions. They are key ...

JoVE Journal

Immunology and Infection

Polarization of M1 and M2 Human Monocyte-Derived Cells and Analysis with Flow Cytometry upon Mycobacterium tuberculosis Infection

Human macrophages are primary host cells of intracellular Mycobacterium tuberculosis (Mtb) infection and thus have a central role in immune control of tuberculosis (TB). We have established an experimental protocol to follow immune polarization of myeloid-derived cells into M1 (classically activated) or M2 (alternatively activated) macrophage-like cells through assessment with a 10-color flow cytometry panel that allows visualization and deep-characterization of green-fluorescent-protein (GFP)-labeled Mtb in diverse macrophages subsets. Monocytes obtained from healthy blood donors were polarized into M1 or M2 cells using differentiation with granulocyte macrophage-colony-stimulating factor (GM-CSF) or macrophage-colony stimulating factor (M-CSF) followed by polarization with IFN-&#947; and lipopolysaccharide (LPS) or IL-4, respectively. Fully polarized M1 and M2 cells were infected with Mtb-GFP for 4 hours before detached Mtb-infected macrophages were stained with flow cytometry at 4- or 24-hours post-infection. Sample acquisition was performed with flow cytometry and the data was analyzed using a flow cytometry analysis software. Manual gating as well as dimensionality reduction with Uniform Manifold Approximation and Projection (UMAP) and phenograph analysis was performed. This protocol resulted in effective M1/M2 polarization characterized by elevated levels of CD64, CD86, TLR2, HLA-DR and CCR7 on uninfected M1 cells, while uninfected M2 cells exhibited a strong up-regulation of the M2 phenotype markers CD163, CD200R, CD206 and CD80. M1-polarized cells typically contained fewer bacteria compared to M2-polarized cells. Several M1/M2 markers were downregulated after Mtb infection, which suggests that Mtb can modulate macrophage polarization. In addition, 24 different cell clusters of different sizes were found to be uniquely distributed among the M1 and M2 uninfected and Mtb-infected cells at 24-hours post-infection. This M1/M2 flow cytometry protocol could be used as a backbone in Mtb-macrophage research and be adopted for special needs in different areas of research.

Polarization of M1 and M2 Human Monocyte-Derived Cells and Analysis with Flow Cytometry upon Mycobacterium tuberculosis Infection

This protocol provides a method to study Mycobacterium tuberculosis infection in human M1- or M2-polarized macrophages based on differentiation of peripheral-blood-monocytes to macrophage-like cells that are infected with the GFP-labeled virulent strain H37Rv, and analyzed with flow cytometry using a 10-color panel including expression of selected M1/M2 markers.

Human macrophages are primary host cells of intracellular Mycobacterium tuberculosis (Mtb) infection and thus have a central role in immune control of ...

Macrophage Differentiation and Polarization into an M2-Like Phenotype using a Human Monocyte-Like THP-1 Leukemia Cell Line

Tumor-associated macrophages (TAM) can switch their expression and cytokine profile according to external stimuli. This remarkable plasticity enables TAM to adapt to ongoing changes within the tumor microenvironment. Macrophages can have either primarily pro-inflammatory (M1-like) or anti-inflammatory (M2-like) attributes and can continually switch between these two main states. M2-like macrophages within the tumor environment are associated with cancer progression and poor prognosis in several types of cancer. Many different methods for inducing differentiation and polarization of THP-1 cells are used to investigate cellular and intercellular mechanisms and the effects of TAM within the microenvironment of tumors. Currently, there is no established model for M2-like macrophage polarization using the THP-1 cell line, and the results of expression and cytokine profiles of macrophages due to certain in vitro stimuli vary between studies. This protocol serves as detailed guidance to differentiate THP-1 monocyte-like cells into M0 macrophages and to further polarize cells into an M2-like phenotype within 14 days. We demonstrate the morphological changes of THP-1 monocyte-like cells, differentiated macrophages, and polarized M2-like macrophages using light microscopy. This model is the basis for cell line models investigating the anti-inflammatory effects of TAM and their interactions with other cell populations of the tumor microenvironment.

M2-like tumor-associated macrophages (TAM) are associated with tumor progression and poor prognosis in cancer. This protocol serves as a detailed guide to reproducibly differentiate and polarize THP-1 monocyte-like cells into M2-like macrophages within 14 days. This model is the basis to investigate the anti-inflammatory effects of TAM within the tumor microenvironment.

Tumor-associated macrophages (TAM) can switch their expression and cytokine profile according to external stimuli. This remarkable plasticity enables TAM ...

In Vitro Culture and Differentiation of Primary Monocytes into Macrophages

Transcript

In Vitro Culture and Differentiation of Primary Monocytes into Macrophages

Production and Characterization of Human Macrophages from Pluripotent Stem Cells

Polarization of M1 and M2 Human Monocyte-Derived Cells and Analysis with Flow Cytometry upon Mycobacterium tuberculosis Infection

Macrophage Differentiation and Polarization into an M2-Like Phenotype using a Human Monocyte-Like THP-1 Leukemia Cell Line