The Price Institute of Surgical Research, University of Louisville, is financially supported by the John W. Price and Barbara Thruston Atwood Price Trust. The funding sources had no role in the design and conduct of the study as well as in the collection, management, analysis, and interpretation of the data.

NOTE: An overview of the steps described in this protocol is shown in Figure 1. The human monocyte-like leukemia cell line THP-1 was purchased. Short tandem repeat analysis was performed to authenticate the THP-1 cell line. Perform all steps under sterile conditions. The THP-1 monocytic cell line grows in suspension and does not attach to cell culture surfaces. Adherence can be induced by differentiating monocytes into macrophage-like cells through, e.g., mechanical stress or specific treatment with PMA.
1. Culturing and maintenance of THP-1 monocyte-like cells
<ol>
	<li>Set a timer for 150 s. Remove the frozen vial containing the THP-1 cell line (Table of Materials) from the liquid nitrogen and thaw it immediately in a clean water bath (37 &#176;C). Start the timer as soon as the vial is put into the water bath. Loosen the cap to release the pressure that is building up due to the thawing process but ensure that the tube opening does not contact the water, to avoid contamination. The optimal time period for thawing the cells lies between 120-150 s. Continue thawing the cell suspension until an ice chip of the size of about 4 mm is left within the vial; then, proceed to the next step immediately.</li>
	<li>Transfer the liquid phase of the cell suspension to a 15 mL tube containing 9 mL of warm (37 &#176;C) growth media (Table of Materials). Then, transfer 1 mL of the warm medium-cell suspension into the THP-1 vial and back into the 15 mL tube to melt the remaining ice chip and flush the vial to assure that no cells are left behind.</li>
	<li>Mix the suspension gently by pipetting up and down with a 1000 &#181;L pipette. Remove a small sample (approximately 10 &#181;L) to count the cells for viability (using trypan blue for exclusion) while they are spun. Spin down the warm cell suspension at 200 x g for 7 min at 37 &#176;C.</li>
	<li>Remove the supernatant completely and resuspend with a certain volume of warm growth medium to achieve a cell density of 5 x 105/mL. Mix the suspension gently and transfer 22 mL of volume into T-75 cell culture flasks (Table of Materials). Store flasks upright in an incubator at 37 &#176;C with 5% carbon dioxide (CO2) concentration. Exchange the growth media every 3-4 days.</li>
</ol>
2. Seeding of THP-1 cells and differentiation into M0 macrophages
<ol>
	<li>Prepare the cell containing growth medium with the respective cell density to seed cells at a density of 3 x 105/mL/well into 24-well cell culture plates (Table of Materials). Mix the medium gently and prepare aliquots of 26 mL, each put into a 50 mL tube. Use each 26 mL aliquot for seeding the cells into a respective plate.</li>
	<li>Transfer 1 mL of the cell-containing medium into each well of a 24-well plate. Mix the media gently by pipetting up and down between transfers.</li>
	<li>Prepare a stock solution of PMA (dissolve 1 mg of PMA in 100 &#181;L of Dimethyl Sulfoxide (DMSO) = ~16 mM solution of PMA in DMSO) and dilute it with cold Phosphate Buffered Saline (PBS) to a final working concentration of 10 ng/&#181;L right before cell treatment (Table of Materials). Keep the solution on ice and use it immediately. Do not refreeze. Add 100 ng of PMA per well. Let each cell plate sit in the incubator without any further treatment for 72 h.</li>
	<li>After 72 h, remove the growth medium and replace it with 1 mL of fresh growth medium. Do not touch the bottom of the wells with pipette tips. Let the cells rest for another 96 h in the incubator.</li>
	<li>After 96 h, repeat step 2.4 (media change) and let the cells rest for another 24 h. 
	&#8203;NOTE: The M0 macrophages are now ready to be used for experiments (Figure 2). Immediately prior to treating the cells as part of further experiments, consider a media change with RPMI only (Table of Materials) since growth media supplements can cause interference with reagents that are added for cell treatment. In case M2-like macrophages are needed, proceed with section 3.</li>
</ol>
3. Polarization of M0 macrophages into M2-like macrophages
<ol>
	<li>Prepare a stock solution of IL-4 and IL-13 (dissolve 20 &#181;g of IL-4 or IL-13 in 200 &#181;L of nuclease-free water) and dilute it to a final working concentration of 2 ng/&#181;L with PBS immediately prior to cell treatment. Keep the solution on ice and use it immediately. Do not refreeze.</li>
	<li>Remove the growth medium and replace it with 1 mL of fresh growth medium. Add 20 ng of interleukin 4 (IL-4) and 20 ng of interleukin 13 (IL-13) per well. Let the cells rest for 48 h in the incubator.</li>
	<li>After 48 h, repeat step 3.2. Let the cells rest for another 48 h in the incubator.</li>
	<li>Remove the growth medium and replace it with 1 mL of fresh growth medium. Let the cells rest for 48 h in the incubator. 
	NOTE: M2-like macrophages are now ready to be used for experiments (Figure 2). Immediately prior to treating the cells as part of further experiments, consider a media change with RPMI only (Table of Materials) since growth media supplements can cause interference.</li>
</ol>
4. Detaching and harvesting macrophages for flow cytometry
NOTE: Use a mechanical method combining cold shocking and cell scraping to detach and harvest the polarized macrophages from plates for flow cytometry.
<ol>
	<li>Remove the warm cell medium and replace it with a mixture of ice-cold PBS (without calcium and magnesium) and 5% fetal bovine serum (FBS), 1 mL per well. Immediately after this, place the cell plate on ice for 45 min. Do not place the cell plate on ice before the warm cell medium is removed, since this will decrease cell viability significantly. Keep the cells on ice only after inducing cold shock with ice-cold PBS/5% FBS mixture.</li>
	<li>After 45 min on ice, scrape off the cells using mini cell scrapers (Table of Materials). Gently transfer the detached macrophages in cold PBS/5% FBS into a 15 mL tube. Keep the tube on ice at all times until cells are stained. 
	NOTE: Pool eight wells of cells to reach adequate cell counts for staining.</li>
</ol>

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	<li>Zhang, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer-associated+fibroblasts+enhance+tumor-associated+macrophages+enrichment+and+suppress+NK+cells+function+in+colorectal+cancer.">Cancer-associated fibroblasts enhance tumor-associated macrophages enrichment and suppress NK cells function in colorectal cancer.</a> Cell Death and Disease. 10 (4), 273 (2019).</li><li>Wang, J., Li, D., Cang, H., Guo, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crosstalk+between+cancer+and+immune+cells:+Role+of+tumor-associated+macrophages+in+the+tumor+microenvironment.">Crosstalk between cancer and immune cells: Role of tumor-associated macrophages in the tumor microenvironment.</a> Cancer Medicine. 8 (10), 4709-4721 (2019).</li><li>Soncin, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tumor+microenvironment+creates+a+niche+for+the+self-renewal+of+tumor-promoting+macrophages+in+colon+adenoma.">The tumor microenvironment creates a niche for the self-renewal of tumor-promoting macrophages in colon adenoma.</a> Nature Communications. 9 (1), 582 (2018).</li><li>Mosser, D. 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The authors declare no potential conflicts of interest.

This protocol on differentiating and polarizing THP-1 monocyte-like cells within 14 days provides a method to obtain macrophages with a distinct M2-like phenotype due to long treatment incubation of cells with adequate resting periods between steps.
Certain steps are critical to this protocol. The doubling time of THP-1 monocytes is approximately 26 h. Cells can be split at a cell density of 9 x 105/mL and should be seeded at a density of 3 x 105/mL during every split. Th...

Tumor-associated macrophages (TAM) and their role in chronic inflammation, the onset of cancer, and tumor development are important targets in recent research1,2. Peripheral blood monocytes that are recruited to the tissue microenvironment of the developing tumor differentiate into macrophages and can be polarized into two main subtypes of macrophages3. The classically activated macrophage represents the primarily pro-inflammatory M1-like phenotype and the alternatively activated M2-like subtype shows predominantly anti-inflammatory characteristics4. Macrophages can switch dynamically between these two main phenotypes depending upon their cellular metabolism, with intermediate subtypes having both inflammatory and anti-inflammatory attributes5. TAM represents a heterogeneous population of both phenotypes. A tumor-promoting function and poor prognosis in different types of cancers is, however, particularly associated with M2-like macrophages6,7,8.
The functional profiles of macrophages and their interaction with other cells within the tumor microenvironment are complex and challenging to capture in a continuously changing environment during ongoing tumor development. Cell lines can provide a homogenous cell population with stable viability in culture, which can facilitate the process of demonstrating defined cellular and intercellular mechanisms. The monocyte-like THP-1 cell line is a legitimate model system for primary human monocytes9. This spontaneously immortalized cell line has been obtained from the peripheral blood of a one-year-old infant with acute monocytic leukemia9,10. The differentiation and polarization of THP-1 cells have been reported by several studies and have been performed in multiple different ways11,12,13,14. Activation and, therefore, the polarization of macrophages into an M1-like phenotype is followed by a compensatory anti-inflammatory rebound mechanism, promoting an M2-like phenotype through cytokines produced by inflammatory macrophages, such as interleukin 6 (IL-6) or itaconate15,16. This might serve as a break mechanism to attenuate an overshooting inflammatory response following cell activation17. The process of differentiating and polarizing monocytes and THP-1 monocyte-like cells into an anti-inflammatory M2-like phenotype is itself also accompanied by pro-inflammatory stimuli that must be overcome. An inflammatory cytokine response can be caused by mechanical stress18, such as changing media to refeed the cells, or adding chemical compounds to differentiate THP-1 cells, such as phorbol&#160;12-myristate 13-acetate (PMA), and induce production of tumor necrosis factor &#945; (TNF&#945;), interleukin 1&#946; (IL-1&#946;) or IL-619. This altered cytokine expression profile as a response to PMA can affect and prevent subsequent macrophage polarization20. Adequate resting periods, as reported before after PMA treatment, allow these inflammatory responses to decrease and facilitate cell polarization into a distinct M2-like phenotype21.
This protocol demonstrates a method to differentiate and polarize THP-1 monocyte-like cells into an M2-like phenotype of macrophages within 14 days.

<table><tbody><tr><td>0.4% trypan blue</td><td>VWR, Radnor, USA</td><td>152-5061</td><td></td></tr><tr><td>1.5 mL microcentrifuge tube</td><td>USA Scientific, Ocala, USA</td><td>1615-5510</td><td></td></tr><tr><td>10 mL serological pipet</td><td>VWR, Radnor, USA</td><td>&amp;nbsp;89130-898</td><td></td></tr><tr><td>1000 &amp;mu;L TipOne pipet tips</td><td>USA Scientific, Ocala, USA</td><td>1111-2821</td><td></td></tr><tr><td>15 mL&amp;nbsp; Centrifuge tube</td><td>VWR, Radnor, USA</td><td>89039-664</td><td></td></tr><tr><td>20 &amp;mu;L TipOne pipet tips</td><td>USA Scientific, Ocala, USA</td><td>1120-1810</td><td></td></tr><tr><td>200 &amp;mu;L TipOne pipet tips</td><td>USA Scientific, Ocala, USA</td><td>1120-8810</td><td></td></tr><tr><td>25 mL serological pipet</td><td>VWR, Radnor, USA</td><td>&amp;nbsp;89130-900</td><td></td></tr><tr><td>5 mL serological pipet</td><td>VWR, Radnor, USA</td><td>&amp;nbsp;89130-896</td><td></td></tr><tr><td>50 mL Centrifuge tube</td><td>VWR, Radnor, USA</td><td>89039-662</td><td></td></tr><tr><td>Accutase solution 500 mL</td><td>Sigma, St. Louis, USA</td><td>A6964</td><td></td></tr><tr><td>Antibiotic Antimycotic Solution (100x), stabilized</td><td>Sigma, St. Louis, USA</td><td>A5955-100 mL</td><td>with 10,000 units penicillin, 10 mg of streptomycin and 25 &amp;mu;g of amphotericin B per mL, sterile-filtered, BioReagent, suitable for cell culture</td></tr><tr><td>Binder CO2 Incubator</td><td>VWR, Radnor, USA</td><td>C170-ULE3</td><td></td></tr><tr><td>CytoOne T-75cm flask with filter cap</td><td>USA Scientific, Ocala, USA</td><td>CC7682-4875</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline (PBS)</td><td>Sigma, St. Louis, USA</td><td>D8537-500 mL</td><td>PBS without calcium chloride and magnesium chloride should be used, since both can alter macrophage polarization</td></tr><tr><td>Eppendorf Centrifuge 5804 R (refrigerated)</td><td>Eppendorf, Enfield, USA</td><td>-</td><td></td></tr><tr><td>Ethyl alcohol (70%)</td><td>-</td><td>-</td><td></td></tr><tr><td>FACSCalibur flow cytometer</td><td>BD Biosciences, San Diego, USA</td><td>-</td><td>The flow cytometer operates with CellQuest software (BD Biosciences)</td></tr><tr><td>Falcon 24-well plate</td><td>VWR, Radnor, USA</td><td>353504</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>ATCC, Manassas, USA</td><td>30-2020</td><td></td></tr><tr><td>FITC Mouse Anti-Human CD14</td><td>BD Biosciences, San Diego, USA</td><td>555397</td><td>Flow cytometry, myeloid cell marker (100 tests)</td></tr><tr><td>FITC Mouse Anti-Human CD80</td><td>BD Pharmingen, San Diego, USA</td><td>557226</td><td>Flow cytometry, M1 marker (100 tests)</td></tr><tr><td>FITC Mouse IgG1 &amp;kappa; Isotype Control</td><td>BD Pharmingen, San Diego, USA</td><td>555748</td><td>Flow cytometry, isotype control for CD80 (100 tests)</td></tr><tr><td>FITC Mouse IgG2a, &amp;kappa; Isotype Control</td><td>BD Biosciences, San Diego, USA</td><td>553456</td><td>Flow cytometry, isotype control for CD14 (100 tests)</td></tr><tr><td>Human BD Fc Block</td><td>BD Biosciences, San Diego, USA</td><td>564220</td><td>Flow cytometry, Fc block (0.25 mg)</td></tr><tr><td>Human interleukin 13 (IL-13)</td><td>R&amp;amp;D, Minneapolis, USA</td><td>IL-771-10 &amp;mu;g</td><td></td></tr><tr><td>Human interleukin 4 (IL-4)</td><td>R&amp;amp;D, Minneapolis, USA</td><td>SRP3093-20 &amp;mu;g</td><td></td></tr><tr><td>Labconco Biosafety Cabinet (Delta Series 36212/36213)</td><td>Labconco, Kansas City, USA</td><td>-</td><td></td></tr><tr><td>L-Glutamine Solution, 200 mM</td><td>ATCC, Manassas, USA</td><td>30-2214</td><td></td></tr><tr><td>Lipopolysaccharide (LPS) from E. coli 0111:B4</td><td>Sigma, St. Louis, USA</td><td>L2630-100 mg</td><td></td></tr><tr><td>Mini Cell Scrapers</td><td>Biotium, Fremont, USA</td><td>22003</td><td></td></tr><tr><td>Neubauer hemocytometer</td><td>Fisher Scientific, Waltham, USA</td><td>02-671-5</td><td></td></tr><tr><td>Nikon Eclipse inverted microscope TS100</td><td>Nikon, Melville, USA</td><td>-</td><td></td></tr><tr><td>Nuclease-free water</td><td>Invitrogen, Carlsbad, USA</td><td>AM9937</td><td></td></tr><tr><td>Olympus Light Microscope RH-2</td><td>Microscope Central, Feasterville, USA</td><td>40888</td><td></td></tr><tr><td>P10 variable pipet- Gilson</td><td>VWR, Radnor, USA</td><td>76180-014</td><td></td></tr><tr><td>P1000 variable pipet-Gilson</td><td>VWR, Radnor, USA</td><td>76177-990</td><td></td></tr><tr><td>P200 variable pipet- Gilson</td><td>VWR, Radnor, USA</td><td>76177-988</td><td></td></tr><tr><td>PE Mouse Anti-Human CD11b</td><td>BD Biosciences, San Diego, USA</td><td>555388</td><td>Flow cytometry, myeloid cell marker (100 tests)</td></tr><tr><td>PE Mouse IgG1, &amp;kappa; Isotype Control</td><td>BD Biosciences, San Diego, USA</td><td>555749</td><td>Flow cytometry, isotype control for CD11b (100 tests)</td></tr><tr><td>PE-Cy 5 Mouse Anti-Human CD206</td><td>BD Pharmingen, San Diego, USA</td><td>551136</td><td>Flow cytometry, M2 marker (100 tests)</td></tr><tr><td>PE-Cy 5 Mouse IgG1 &amp;kappa; Isotype Control</td><td>BD Pharmingen, San Diego, USA</td><td>555750</td><td>Flow cytometry, isotype control for CD206 (100 tests)</td></tr><tr><td>Phorbol 12-myristate 13-acetate (PMA)</td><td>Sigma, St. Louis, USA</td><td>P8139</td><td></td></tr><tr><td>Powerpette Plus pipettor</td><td>VWR, Radnor, USA</td><td>75856-448</td><td></td></tr><tr><td>Precision Water bath (model 183)</td><td>Precision Scientific, Chicago, USA</td><td>66551</td><td></td></tr><tr><td>RPMI-1640 Medium</td><td>ATCC, Manassas, USA</td><td>30-2001</td><td></td></tr><tr><td>THP-1 cell line, American Type Culture Collection (ATCC)</td><td>ATCC, Manassas, USA</td><td>TIB-202</td><td></td></tr></tbody></table>

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 macrophages were characterized, and M2-polarization was validated using flow cytometry for Cluster of Differentiation markers (CD) CD14, CD11b, CD80 (M1-like marker), and CD206 (M2-like marker). Flow cytometry staining was performed according to the manufacturer's instructions. Macrophages were washed with PBS/5% FBS and incubated with Fcγ-receptor block to avoid unspecific binding. Cells were then stained with FITC-conjugated mouse anti-human CD14 and CD80 antibodies, with PE-conjugated mouse anti-human CD11b a...

Watch this Scientific Journal Video about Macrophage Differentiation and Polarization into an M2-Like Phenotype using a Human Monocyte-Like THP-1 Leukemia Cell Line at JoVE.com

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

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 ...

macrophage-differentiation-polarization-into-an-m2-like-phenotype

Research

JoVE Journal

Immunology and Infection

27.1K Views.  University of Louisville. Ontstekingsremmende tumor geassocieerde macrofagen zijn gekoppeld aan progressie en slechte prognose bij kanker. Dit protocol is een gids om THP-1 monocytachtige cellen binnen 14 dagen te differentiëren en te polariseren in M2-achtige macrofagen. Momenteel is er geen gevestigd model voor ontstekingsremmende THP-1 polarisatie.Dit protocol maakt gebruik van adequate rustperioden en een langdurig polarisatieproces om een duidelijk M2-achtig macrofaagsubtype te creëren. Tumorontwikkeling...

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

Proteomic Analysis of Human Macrophage Polarization Under a Low Oxygen Environment

Macrophages are innate immune cells involved in a number of physiological functions ranging from responses to infectious pathogens to tissue homeostasis. The various functions of these cells are related to their activation states, which is also called polarization. The precise molecular description of these various polarizations is a priority in the field of macrophage biology. It is currently acknowledged that a multidimensional approach is necessary to describe how polarization is controlled by environmental signals. In this report, we describe a protocol designed to obtain the proteomic signature of various polarizations in human macrophages. This protocol is based on a label-free quantification of macrophage protein expression obtained from in-gel fractionated and Lys C/trypsin-digested cellular lysis content. We also provide a protocol based on in-solution digestion and isoelectric focusing fractionation to use as an alternative. Because oxygen concentration is a relevant environmental parameter in tissues, we use this protocol to explore how atmospheric composition or a low oxygen environment affects the classification of macrophage polarization.

We present a protocol to obtain proteomic signatures of human macrophages and apply this to determination of the impact of a low oxygen environment on macrophage polarization.

Macrophages are innate immune cells involved in a number of physiological functions ranging from responses to infectious pathogens to tissue homeostasis. ...

Biochemistry

Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic ...

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the phenotypic and functional differences between murine and human monocyte-derived macrophages. Therefore, we here describe an in vitro model to generate and study primary human macrophages. Briefly, after density gradient centrifugation of peripheral blood drawn from a forearm vein, monocytes are isolated from peripheral blood mononuclear cells using negative magnetic bead isolation. These monocytes are then cultured for six days under specific conditions to induce different types of macrophage differentiation or polarization. The model is easy to use and circumvents the problems caused by species-specific differences between mouse and man. Furthermore, it is closer to the in vivo conditions than the use of immortalized cell lines. In conclusion, the model described here is suitable to study macrophage biology, identify disease mechanisms and novel therapeutic targets. Even though not fully replacing experiments with animals or human tissues obtained post mortem, the model described here allows identification and validation of disease mechanisms and therapeutic targets that may be highly relevant to various human diseases.

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages are important cells of the innate immune system. Here, we describe an easy to use in vitro model to generate these cells. Using gradient centrifugation, negative bead isolation and specific cell culture conditions, monocyte-derived macrophages can be generated for phenotypic and functional studies.

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

Isolation of Human Monocytes by Double Gradient Centrifugation and Their Differentiation to Macrophages in Teflon-coated Cell Culture Bags

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to understand the underlying mechanisms of these diseases. We therefore present a straightforward protocol for the isolation of human monocytes from buffy coats, followed by a differentiation procedure which results in high macrophage yields. The technique relies mostly on commonly available lab equipment and thus provides a cost and time effective way to obtain large quantities of human macrophages. Briefly, buffy coats from healthy blood donors are subjected to a double density gradient centrifugation to harvest monocytes from the peripheral blood. These monocytes are then cultured in fluorinated ethylene propylene (FEP) Teflon-coated cell culture bags in the presence of macrophage colony-stimulating factor (M-CSF). The differentiated macrophages can be easily harvested and used for subsequent studies and functional assays. Important methods for quality control and validation of the isolation and differentiation steps will be highlighted within the protocol. In summary, the protocol described here enables scientists to routinely and reproducibly isolate human macrophages without the need for cost intensive tools. Furthermore, disease models can be studied in a syngeneic human system circumventing the use of murine macrophages.

We present a simple and efficient protocol for the generation of human macrophages. Buffy coats are processed by double density gradient centrifugation and isolated monocytes are then differentiated to macrophages in Teflon-coated cell culture bags. This maximizes macrophage yields and facilitates cell harvesting for subsequent experiments.

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to ...

Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation of experiments, fresh or frozen monocytes are cultured in flasks for 1 week with M-CSF to induce their differentiation into macrophages. Then, the macrophages can be harvested and seeded into culture wells at required cell densities for carrying out experiments. The use of defined numbers of macrophages rather than defined numbers of monocytes to initiate macrophage cultures for experiments yields macrophage cultures in which the desired cell density can be more consistently attained. Use of cryopreserved monocytes reduces dependency on donor availability and produces more homogeneous macrophage cultures.

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. The protocol utilizes cryopreservation of monocytes coupled with their bulk differentiation into macrophages. Then harvested macrophages can then be seeded into culture wells at required cell densities for carrying out experiments.

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation ...

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 ...

Assessing Immunomodulatory Effects of Implant Surfaces on Macrophages

Polarization and Characterization of M1 and M2 Human Monocyte-Derived Macrophages on Implant Surfaces 

Foreign body reaction (FBR), an immune-mediated complex healing process, plays a crucial role in integrating implants into the body. Macrophages, as the first line of immune system interaction with implant surfaces, play a bidirectional role in modulating the inflammation-regeneration balance. For a deep understanding and the evaluation of the reactions between implant materials and immune responses, reliable in vitro methods and protocols are pivotal. Among different in vitro models, primary monocyte-derived macrophages (MDMs) present an excellent model for investigating macrophage-implant interactions. We have implemented an experimental protocol to evaluate the polarization of MDMs into M1 (classically activated) and M2 (alternatively activated) macrophages on implant surfaces. We isolated blood monocytes from healthy donors and differentiated them into macrophages using macrophage colony-stimulating factor (M-CSF). Differentiated macrophages were cultured on implant surfaces and polarized into M1 and M2 subtypes. M1 polarization was achieved in the presence of interferon (IFN)-&#947; and lipopolysaccharide (LPS), while M2 polarization was performed in a medium containing interleukin (IL)-4 and IL-13. We evaluated macrophage phenotypes by Enzyme-linked&#160;Immunosorbent Assay (ELISA), confocal laser scanning microscopy (CLSM), and quantitative real-time PCR (qRT-PCR) based on panels of secreted cytokines, cell surface markers, and expressed genes. The extracted RNA was transformed into complementary DNA (cDNA), and qRT-PCR was used to quantify mRNA related to M1 and M2 macrophages. Accordingly, M1 macrophages have been characterized by higher expression of proinflammatory Tumor necrosis factor (TNF-&#945;) cytokine and CCR7 surface marker compared to M2&#160;macrophages, which exhibited higher levels of CD209 and CCL13. Consequently, CCR7 and CD209 were identified as specific and reliable markers of M1 and M2 macrophage subtypes by immunostaining and visualizing by CLSM. Further confirmation was achieved by ELISA detecting elevated TNF-&#593; level in M1 and increased CCL13 in M2 cells. The proposed markers and experimental setup can be used effectively to assess the immunomodulatory potential of implants.

Polarization and Characterization of M1 and M2 Human Monocyte-Derived Macrophages on Implant Surfaces

Here, we present a detailed protocol for assessing the immunomodulatory potential of implant surfaces in vitro, aiming to improve the reliability and reproducibility of current protocols and promoting further research. Secretory cytokine profiles, mRNA expression, and cell surface markers were monitored using blood monocyte-derived macrophages to investigate macrophage polarization cultivated on titanium.

Foreign body reaction (FBR), an immune-mediated complex healing process, plays a crucial role in integrating implants into the body. Macrophages, as the ...

Prediction of Red Blood Cell Antibody Significance Using the Monocyte-Macrophage Assay

Derived from monocytes in the bone marrow, macrophages are large, innate immune cells that play a major role in clearing dead cells, debris, tumor cells, and foreign pathogens. The phagocytic capacity of monocytes versus macrophages is a concept that is not well understood. Here, we aim to examine a difference in the phagocytosis of monocytes versus macrophages, specifically M1/M2 macrophages, against various opsonized red cells using a modified and updated version of the established monocyte monolayer assay (MMA). Peripheral blood mononuclear cells (PBMCs) were isolated from donor buffy coats. Using purified monocytes, inflammatory M1 and anti-inflammatory M2 macrophages were produced by in vitro culture and polarization. M1/M2 cells were harvested and used in an MMA-like assay, which we refer to as the M-MA, to decipher clinically significant phagocytosis of various red cell antibodies. A phagocytic index (PI) &#62; 5 was deemed clinically significant phagocytosis with the use of monocytes. A phagocytic index (PI) &#62; 12 was deemed clinically significant phagocytosis with the use of M1/M2 macrophages. M2 macrophages demonstrate an increased ability to phagocytose opsonized RBCs compared to monocytes and M1s. The same weak antibody (anti-S) yields significant phagocytosis with only M2 macrophages (PI=43) but not M1s (PI=2) or monocytes (PI=0), and this was demonstrated repeatedly using various antibodies. The use of M2 macrophages instead of monocytes may allow for more accurate results as these cells are more phagocytic, offering further clinical relevance to the assay. Further studies with different antibodies to red blood cells, including validation of the monocyte-macrophage assay (M-MA) with antibodies having known clinical significance, may show the M-MA&#160;more useful to help predict clinically significant red cell alloantibodies and transfusion reactions. This method will advance the field of transfusion medicine and immunology.

We have developed enhancements and updated methods for the existing monocyte-monolayer assay (MMA), in which macrophages are used to help better predict the clinical relevance of red cell alloantibodies in transfusion medicine and immunology. This assay is named the monocyte-macrophage assay (M-MA).

Derived from monocytes in the bone marrow, macrophages are large, innate immune cells that play a major role in clearing dead cells, debris, tumor cells, ...

Reprograming Model of Human Monocyte-derived Macrophages for In-vitro Assays

Cells of the monocyte-macrophage lineage are multifunctional and found in almost all body tissues. They coordinate innate and adaptive immunity&#180;s initiation and resolution phases, significantly affecting protective immunity and immune-mediated pathological injury. While tissue-resident macrophages are key players in maintaining homeostasis in a steady state, large amounts of monocytes are recruited from the peripheral blood into the tissue following damage or inflammatory insults. Monocyte-derived macrophages (M-DM) can differentiate into many dynamic subtypes, and their phenotypes and functions depend on the local tissue environment. 
To compare different stimuli or environmental conditions during M-DM differentiation and polarization, we standardized an in-vitro model of human nonpolarized M-DM M0 and some cardinal cytokine-polarized macrophages, IFN&#947;/LPS-derived M1, IL-4-derived M2a, and IL-10 or dexamethasone-derived M2c, to analyze skewing reprogramming of M-DM by flow cytometry and real-time PCR. We found that CD64, CD206, CD163, CD14, and MERTK can clearly discriminate unpolarized M0 and polarized M1, M2a, and M2c by flow cytometry. Moreover, we defined IRF1 and CXCL10 as specific genes for classical IFN&#947;/LPS-derived M1-, IRF4-, CCL22-, and TGM2-specific transcripts for IL-4-derived M2a, and the MERTK gene for dexamethasone-derived M2c. To summarize, our standardized M-DM protocol could give the cardinal in-vitro map to analyze the differentiation and polarization of human M-DM under diverse stimuli.

Monocytes and macrophages are very plastic and reprogrammable immune cells crucial for health and disease. They sense and respond to a broad range of stimuli by adopting specific differentiation programs and phenotypes. We have standardized an in-vitro model to study macrophage polarization and reprogramming, providing a valuable tool for research.

Cells of the monocyte-macrophage lineage are multifunctional and found in almost all body tissues. They coordinate innate and adaptive immunity&#180;s ...

A Fully Differentiated Macrophage Infection Model of Monocyte-Derived Cells with Mycobacterium tuberculosis

Source: Mily, A., et al. <a href="https://app.jove.com/v/61807">Polarization of M1 and M2 Human Monocyte-Derived Cells and Analysis with Flow Cytometry upon Mycobacterium tuberculosis Infection.</a> J. Vis. Exp.(2020).
This video demonstrates a method to study the responses to Mycobacterium tuberculosis infection in human M1 and M2 polarized monocyte-derived macrophages. The use of flow cytometry enables the study of a variety of surface markers associated with infected and uninfected macrophage populations.

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

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