The Department of Transfusion Medicine, Clinical Center, National Institutes of Health, provided elutriated monocytes. This work was supported by the Intramural Research Program, National Heart, Lung, and Blood Institute, National Institutes of Health.

백혈구 성분 채집은 보건 기관 검토위원회의 국립 연구소의 승인을 인간 대상의 연구 프로토콜에 따라 수행 하였다. 1. 분리 및 단핵구의 냉동 보존 <ol><li> 인간의 기증자의 백혈구 성분 채집하여 단핵 세포를 확보하고, 참고 문헌 7,8에 설명 된대로 단핵 세포의 연속 역류 원심 분리 세광에 의해 단핵구을 풍부. 제조자가 지정한 세광 버퍼 - (90 % 단핵구 약 80) 약 100 × 106 씻겨져 걸러진 세포를 얻습니다. 혈구를 사용하여 세포를 계산합니다. </li><li> 300 XG에 15 ml의 폴리 프로필렌 튜브에 원심 분리기 단핵구 실온에서 5 분간. </li><li> 최종 90 % FBS / 10 % 디메틸 술폭 시드 농도 50 × 106 세포 / ml를 달성하는 디메틸 술폭 시드 이어 FBS에서 세포를 재현 탁 조심스럽게 상층 액을 제거하고. </li><li> 세포 현탁액의 t의 각 ML을 추가개인 cryovial을 오. </li><li> 셀 냉동 컨테이너에 배치 크리오 바이알 (cryovial)로 전송하고 -80 ° C의 냉동고에 24 시간 동안의 장기 저장을 위해 액체 질소 cryovial 저장 탱크로 전환하기 전에. </li></ol> 대 식세포에 단핵구 2. 차별화 <ol><li> 빠르게 37 ℃의 수조에 전달하고, 세포 현탁액은 약 70 % 융해되었을 때 즉시 제거하여 세포를 해동 cryovial. </li><li> 즉시 실온에서 완전 해동시에 C가 로스웰 파크 메모리얼 연구소 (RPMI) 2 mM L- 글루타민, 50 NG / ㎖ M-CSF, 25 ng의 / ㎖와 1640 가온 37 50 ㎖로 한 용액을 cryovial 내용을 전송 인터루킨 10 (IL-10) 및 10 % FBS (완전 배지). </li><li> 전송이 75cm이 플라스틱 세포 배양 플라스크 각각에 단구 현탁액 25 ㎖. </li><li> 48 시간 동안 5 % CO 95분의 2 %의 공기와 37 ℃ 세포 배양 인큐베이터에서 배양 물을 인큐베이션. </li><li> CU를 씻어RPMI 1640 배지를 부드럽게 느슨하게 붙어있는 세포를 빠지 않도록 배지를 제거하고 (37 ℃로 미리 가온) 10 mL로 3 회 ltures. </li><li> 세정 후, 단핵 세포가 분화 될 때까지 중간마다 이일을 변경, 신선한 완전 배지를 추가 합류가 충분히 증식. 이것은 문화의 약 1 주일이 필요합니다. </li></ol> 3. 수확 식세포는 실험을 시작합니다 <ol><li> 10 ㎖의 플라스크에 3 회 분화 된 대 식세포 린스 예열 37 ° C, 0.25 % 트립신 에틸렌 디아민 테트라 아세트산 (EDTA) 용액 10 ㎖를 첨가하기 전에 2 + Mg를 칼슘 2 않고 37 ° C 둘 베코 인산염 완충 식염수를 예열. </li><li> 대 식세포의 약 90 %가 반올림과 분리해야하는 시간에 15 분 - 10 세포 배양 인큐베이터에 넣어 플라스크. 플라스크의 현미경 검사하여이를 확인합니다. </li><li> 10 % FBS를 포함 RMPI 1640 배지 10 ㎖의에 추가상단 트립신. </li><li> 5 분 동안 원심 분리기, 50 ml의 폴리 프로필렌 튜브에 플라스크에서 300 XG를 대식 세포 현탁액을 전송하고, 상층 액을 버린다. </li><li> 부드럽게 완전 배지 1 ㎖에 재현 탁 식세포. </li><li> 10 ㎕의 트리 판 블루 용액으로 대식 세포 현탁액 10 μL를 혼합하고 혈구를 사용 대식 세포 수 및 생존율 (전형적으로&gt; 95 %)를 결정한다. </li><li> 셀 수에 기초하여 (일반적으로 약 15-18 × 105 식세포) 원하는 시딩 세포 밀도를 달성하기 위해 추가적인 완전 배지를 추가한다. 참고 : 12 웰 플레이트의 웰 당 1.5 ml의 배지에서 1 × 10 5 대 식세포를 거의 합류 문화를 생성합니다. </li><li> 접종 후, 세포 부착을 허용하기 위해 5 % CO 95분의 2 %의 공기와 37 ℃ 세포 배양 인큐베이터에서 밤새 배양 한 대 식세포. 그런 다음, 원하는 실험을 시작한다. </li></ol>

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The authors have nothing to disclose.

정의 식세포 유형을 생성하는 대 식세포 생물학을 연구 할 때 조사자에 의해 얻어진 결과가 충돌의 일부를 명확하게 할 수있다. 다양한 배양 조건 및 분화 인자의 사용은 차 인간 대 식세포는 아주 다른 식세포 타입을 초래할 수 연구자에 의해 인식되지 않을 수있는 점을 생성한다. 예를 들어, 대 식세포는 때때로 어떤 혈청, 인간 혈청 형, FBS 만 M-CSF, 또는 M-CSF 또는 GM-CSF를 함유 FBS 보충 된 인간 혈...

Study of cultured macrophages is a useful model to understand the function of these cells in inflammation such as occurs in atherosclerotic plaques. When the research focus is on human diseases involving macrophages, it is useful to study primary human macrophages rather than non-human macrophages to avoid species differences. Also, the effects of cell transformation can be avoided by using primary macrophages rather than macrophage cell lines. For this purpose, macrophages differentiated from monocytes isolated from human blood serve as a means of obtaining primary human macrophages. 
Tissue macrophages may be either resident within tissues or may be derived from monocytes that migrate into the tissue and differentiate into macrophages 1. Two types of human monocyte-derived macrophages have been defined that differ not only in morphology but also gene expression and cell function 2. These two types are obtained from monocytes that are differentiated into macrophages in the presence of M-CSF + fetal bovine serum (FBS) and monocytes that are differentiated into macrophages in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) + FBS 2-6. In our experience (unpublished observation), use of human serum rather than FBS generates the GM-CSF type regardless of whether M-CSF or GM-CSF is included in the differentiation medium. M-CSF type macrophages tend to be more elongated than GM-CSF type macrophages, which resemble fried eggs in their morphology. Investigators should recognize that human M-CSF and GM-CSF monocyte-derived macrophages are not the same as so called M1 and M2 mouse bone marrow-derived macrophages 6.
During research using human monocyte-derived M-CSF differentiated macrophages, we experienced difficulty related to the availability of monocytes to initiate experiments and variation in the obtained cell densities of macrophages during differentiation of monocytes into macrophages. To overcome these problems, we have developed the following protocol in which monocytes are frozen until required for use, and monocytes are differentiated in bulk into macrophages that can then be removed from culture flasks and plated at desired cell densities to obtain more uniform cultures from experiment to experiment.

<table><tbody><tr><td>Cellbind 12-well culture plate</td><td>Corning</td><td>3336</td><td></td></tr><tr><td>CELLSTAR, T-75 flask, tissue culture treated</td><td>Greiner Bio-One North America</td><td>658157</td><td></td></tr><tr><td>RPMI 1640 culture medium</td><td>Cellgro Mediatech</td><td>15-040-CM</td><td>warmed to 37 &amp;deg;C</td></tr><tr><td>L-Glutamine</td><td>Cellgro Mediatech</td><td>25-005-CI</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Gibco</td><td>16000-036</td><td></td></tr><tr><td>M-CSF</td><td>PeproTech</td><td>300-25</td><td></td></tr><tr><td>GM-CSF</td><td>PeproTech</td><td>300-03</td><td></td></tr><tr><td>IL-10&amp;nbsp;</td><td>PeproTech</td><td>200-10</td><td></td></tr><tr><td>DMSO</td><td>Sigma</td><td>D2650</td><td></td></tr><tr><td>Cryovial&amp;nbsp;</td><td>Thermo Scientific&amp;nbsp;</td><td>375418</td><td></td></tr><tr><td>DPBS without Ca&lt;sup&gt;2+&lt;/sup&gt; and Mg&lt;sup&gt;2+&lt;/sup&gt;&amp;nbsp;</td><td>Corning cellgro</td><td>21-031-CV</td><td></td></tr><tr><td>0.25% Trypsin-EDTA&amp;nbsp;</td><td>Gibco</td><td>25200-056</td><td></td></tr><tr><td>50 ml polypropylene conical tube</td><td>Falcon</td><td>352070</td><td></td></tr><tr><td>Trypan Blue</td><td>Lonza</td><td>17-942E</td><td></td></tr><tr><td>Neubauer-improved bright light hemocytometer</td><td>Paul Marienfeld GmbH &amp;amp; Co. KG</td><td>610031</td><td>http://www.marienfeld-superior.com/index.php/counting-chambers/articles/counting-chambers.html</td></tr><tr><td>CoolCell LX cell freezing container</td><td>BioCision</td><td>BCS-405</td><td>other freezing containers also should&amp;nbsp; be adequate for this step</td></tr><tr><td>Liquid Nitrogen Storage System, CryoPlus 1</td><td>Thermo Scientific&amp;nbsp;</td><td>7400</td><td>any liquid nitrogen tank should be adequate</td></tr></tbody></table>

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.

트리 판 블루 (9) 염색으로 결정 신선 또는 냉동 보존 단핵 세포의 생존 도표 1. 95 % 이상이고 그림 2는 낮은과 높은 배율에서 비교, 각각 식세포에 신선하고 냉동 보관 (즉, 냉동) 단핵구의 분화의 진행. 냉동 보관 단핵구 확산하지만 반올림 남아 있지 않습니다 단핵 세포를 분화의 모집단을 보여와 신선한 비교합니다. 상...

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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. 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.

대 식세포 콜로니 자극 인자 차별화 된 인간 단핵구 유래 대 식세포의 문화

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

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Universidad de Cartagena UDEC

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Immunology and Infection

29.1K 조회수.  National Institutes of Health. 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.

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

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.

<em&gt; 체외</em인간 대식 세포의 분화와 양극화의 이질성을 연구&gt; 모델

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

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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.

인간의 두 그라데이션 원심 분리하여 단핵구와 테프론 코팅 세포 배양 가방에서 대 식세포로 분화의 분리

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

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.

Bone marrow cells cultured with granulocyte macrophage colony stimulating factor (GM-CSF) generate a heterogeneous culture containing macrophages and dendritic cells (DCs). This method highlights using MHCII and hyaluronan (HA) binding to differentiate macrophages from the DCs in the GM-CSF culture. Macrophages in this culture have many similarities to alveolar macrophages.

마우스 골수에서 생성 및 GM-CSF의 식별 파생 폐포와 같은 대 식세포와 수지상 세포

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. ...

Isolation, Transfection, and Culture of Primary Human Monocytes

Human immunodeficiency virus (HIV) remains a major health concern despite the introduction of combined antiretroviral therapy (cART) in the mid-1990s. While antiretroviral therapy efficiently lowers systemic viral load and restores normal CD4+ T cell counts, it does not reconstitute a completely functional immune system. A dysfunctional immune system in HIV-infected individuals undergoing cART may be characterized by immune activation, early aging of immune cells, or persistent inflammation. These conditions, along with comorbid factors associated with HIV infection, add complexity to the disease, which cannot be easily reproduced in cellular and animal models. To investigate the molecular events underlying immune dysfunction in these patients, a system to culture and manipulate human primary monocytes in vitro is presented here. Specifically, the protocol allows for the culture and transfection of primary CD14+ monocytes obtained from HIV-infected individuals undergoing cART as well as from HIV-negative controls. The method involves isolation, culture, and transfection of monocytes and monocyte-derived macrophages. While commercially available kits and reagents are employed, the protocol provides important tips and optimized conditions for successful adherence and transfection of monocytes with miRNA mimics and inhibitors as well as with siRNAs.

Presented here is an optimized protocol for isolating, culturing, transfecting, and differentiating human primary monocytes from HIV-infected individuals and healthy controls.

1차 인간 단핵구의 고립, 형질전환 및 문화

Human immunodeficiency virus (HIV) remains a major health concern despite the introduction of combined antiretroviral therapy (cART) in the mid-1990s. ...

In Vitro Stimulation and Visualization of Extracellular Trap Release in Differentiated Human Monocyte-derived Macrophages

The release of extracellular traps (ETs) by neutrophils has been identified as a contributing factor to the development of diseases related to chronic inflammation. Neutrophil ETs (NETs) consist of a mesh of DNA, histone proteins, and various granule proteins (i.e., myeloperoxidase, elastase, and cathepsin G). Other immune cells, including macrophages, can also produce ETs; however, to what extent this occurs in vivo and whether macrophage extracellular traps (METs) play a role in pathological mechanisms has not been examined in detail. To better understand the role of METs in inflammatory pathologies, a protocol was developed for visualizing MET release from primary human macrophages in vitro, which can also be exploited in immunofluorescence experiments. This allows further characterization of these structures and their comparison to ETs released from neutrophils. Human monocyte-derived macrophages (HMDM) produce METs upon exposure to different inflammatory stimuli following differentiation to the M1 pro-inflammatory phenotype. The release of METs can be visualized by microscopy using a green fluorescent nucleic acid stain that is impermeant to live cells (e.g., SYTOX green). Use of freshly isolated primary macrophages, such as HMDM, is advantageous in modeling in vivo inflammatory events that are relevant to potential clinical applications. This protocol can also be used to study MET release from human monocyte cell lines (e.g., THP-1) following differentiation into macrophages with phorbol myristate acetate or other macrophage cell lines (e.g., the murine macrophage-like J774A.1 cells).

Presented here is a protocol to detect macrophage extracellular trap (MET) production in live cell culture using microscopy and fluorescence staining. This protocol can be further extended to examine specific MET protein markers by immunofluorescence staining.

분화 한 인간 단핵구 유래 대식세포에서 세포 외 트랩 방출의 시험관 내 자극 및 시각화

The release of extracellular traps (ETs) by neutrophils has been identified as a contributing factor to the development of diseases related to chronic ...

Differentiation of Monocytes into Phenotypically Distinct Macrophages After Treatment with Human Cord Blood Stem Cell (CB-SC)-Derived Exosomes

Stem Cell Educator (SCE) therapy is a novel clinical approach for the treatment of type 1 diabetes and other autoimmune diseases. SCE therapy circulates the isolated patient&#8217;s blood mononuclear cells (e.g., lymphocytes and monocytes) through an apheresis machine, co-cultures the patient&#8217;s blood mononuclear cells with adherent cord blood-derived stem cells (CB-SC) in the SCE device, and then returns these &#8220;educated&#8221; immune cells to the patient&#8217;s blood. Exosomes are nano-sized extracellular vesicles between 30&#8210;150 nm existing in all biofluid and cell culture media. To further explore molecular mechanisms underlying SCE therapy and determine the actions of exosomes released from CB-SC, we investigate which cells phagocytize these exosomes during the treatment with CB-SC. By co-culturing Dio-labeled CB-SC-derived exosomes with human peripheral blood mononuclear cells (PBMC), we found that CB-SC-derived exosomes were predominantly taken up by human CD14-positive monocytes, leading to the differentiation of monocytes into type 2 macrophages (M2), with spindle-like morphology and expression of M2-associated surface molecular markers. Here, we present a protocol for the isolation and characterization of CB-SC-derived exosomes and the protocol for the co-culture of CB-SC-derived exosomes with human monocytes and the monitoring of M2 differentiation.

Differentiation of Monocytes into Phenotypically Distinct Macrophages After Treatment with Human Cord Blood Stem Cell CB-SC-Derived Exosomes

Exosome application is an emerging tool for drug development and regenerative medicine. We establish an exosome isolation protocol with high purity to isolate exosomes from novel identified stem cells called CB-SC for mechanistic studies. We also coculture CB-SC-derived exosomes with human monocytes, leading to their differentiation into phenotypically distinct macrophages.

인간 코드 혈액 줄기 세포 (CB-SC)-유래 엑소좀으로 치료 후 페노전성 뚜렷한 대식세포로 단핵구를 분화

Stem Cell Educator (SCE) therapy is a novel clinical approach for the treatment of type 1 diabetes and other autoimmune diseases. SCE therapy circulates ...

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.

인간 단세포와 같은 THP-1 백혈병 세포선을 사용하여 M2와 같은 표현형으로 대식세포 분화 및 편광

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

Isolation and Culture of Bone Marrow-Derived Macrophages from Mice

Macrophages have important effector functions in homeostasis and inflammation. These cells are present in every tissue in the body and have the important ability to change their profile according to the stimuli present in the microenvironment. Cytokines can profoundly affect macrophage physiology, especially IFN-&#947; and interleukin 4, generating M1 and M2 types respectively. Because of the versatility of these cells, the production of a population of bone marrow-derived macrophages can be a basic step in many experimental models of cell biology. The aim of this protocol is to help researchers in the isolation and culture of macrophages derived from bone marrow progenitors. Bone marrow progenitors from pathogen-free C57BL/6 mice are transformed into macrophages upon exposure to macrophage colony-stimulating factor (M-CSF) that, in this protocol, is obtained from the supernatant of the murine fibroblast lineage L-929. After incubation, mature macrophages are available for use from the 7th to the 10th day. A single animal can be the source of approximately 2 x 107 macrophages. Therefore, it is an ideal protocol for obtaining large amounts of primary macrophages using basic methods of cell culture.

The present protocol describes the isolation and culture of bone marrow-derived macrophages from mice.

Macrophages have important effector functions in homeostasis and inflammation. These cells are present in every tissue in the body and have the important ...

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 ...

In Vitro Culture and Differentiation of Primary Monocytes into Macrophages

Source: Plaisance-Bonstaff, K., et al. <a href="https://appjove.unicartagenaproxy.elogim.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).

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