Name: culture of macrophage colony-stimulating factor differentiated human monocyte-derived macrophages
Uploaded: 2016-06-30T15:00:00.000+00:00
Duration: 6 min 46 s
Description: Watch this Scientific Journal Video about Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages at JoVE.com

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.

Lökoferez Sağlık kurumsal inceleme kurulu Ulusal Sağlık Enstitüleri tarafından onaylanmış bir insan öznenin araştırma protokol kapsamında gerçekleştirildi. 1. İzolasyon ve Monositler Kryoprezervasyon <ol><li> İnsan donörlerin lökaferez tarafından mononükleer hücreleri elde, ve referanslar 7,8 açıklandığı gibi mononükleer hücrelerin sürekli karşı akım santrifüj elutrasyonun tarafından monositler zenginleştirmek. Üretici tarafından belirtilen sivili ayrıştırma tamponunda - (% 90 monositler yaklaşık 80) yaklaşık 100 x 10 6 elutriated hücre elde. Bir hemasitometre kullanarak hücreleri saymak. </li><li> 300 xg&#39;de 15 mi polipropilen tüp içinde santrifüj monositler Oda sıcaklığında 5 dakika karıştırıldı. </li><li> Bir son% 90 FBS /% 10 dimetil sülfoksit konsantrasyonu 50 x 10 6 hücre / ml elde etmek için, dimetil sülfoksit, ardından FBS içinde tekrar süspansiyon hücreleri hafifçe Süpernatantı ve. </li><li> Hücre süspansiyonu t her ml ekleyinbağımsız bir cryovial O. </li><li> Hücre dondurma kapta cryovials yerleştir ve transfer -80 ° C sıcaklıkta dondurucuya 24 saat boyunca, uzun süreli depolama için bir sıvı azot depolama tankı cryovial aktarmadan önce. </li></ol> Makrofajlar içine Monositler 2. Farklılaşma <ol><li> hızlı bir şekilde 37 ° C su banyosuna transfer ve hücre süspansiyonu yaklaşık% 70 çözülmüş zaman derhal kaldırarak hücre cryovial çözülme. </li><li> Hemen, oda sıcaklığında komple erime üzerine Cı Roswell Park Memorial Institute (RPMI), 2 mM L-glutamin, 50 ng / ml M-CSF, 25 ng / ml 1640 ortamı ısıtıldı, 37 °, 50 ml 1 ml cryovial içeriğini aktarmak interlökin-10 (IL-10) ve% 10 FBS (tam ortam). </li><li> Aktarım, iki 75 cm 2 Plastik hücre kültürü şişesinden her birine monosit süspansiyonu 25 mi. </li><li> 48 saat boyunca% 5 CO2 /% 95 hava ile 37 ° C hücre kültürü inkübatöründe kültürleri inkübe edin. </li><li> cu durulayınRPMI 1640 ortamı yavaşça bir gevşek bağlı hücrelerin yerini değiştirmemek için, kültür ortamının çıkarılması, (37 ° C önceden ısıtılmış) mi, 10 ile ltures 3 kez. </li><li> durulama sonrasında, monositler ayırt kadar ortam her 2 günde bir değişen, taze tamamlanmış besiyeri ekleyebilir ve konfluent hale gelmeye yeterli çoğalır. Bu kültürün yaklaşık bir hafta gerektirir. </li></ol> 3. Hasat Makrofajlar Deneyler Başlatacak <ol><li> 10 ml bir şişe içinde 3 kez ayırt makrofajlar durulayın önceden ısıtılmış 37 ° C,% 0.25 tripsin etilendiamintetraasetik asit (EDTA) çözeltisi 10 ml ilave edilmeden önce 2 + ve Mg2 +, Ca olmayan 37 ° C Dulbecco fosfat tamponlu tuzlu su önceden ısıtılmış. </li><li> makrofajlar yaklaşık% 90&#39;ı yuvarlak ve müstakil olması gereken zaman 15 dakika - 10 hücre kültürü inkübatör Yeri balon. Şişenin mikroskopik inceleme ile bu doğrulayın. </li><li> % 10 FBS içeren RMPI 1640 ortamında 10 ml ŞÜst trypsinization. </li><li> 5 dakika boyunca santrifüj 50 ml polipropilen tüpe şişeye 300 xg makrofaj hücre süspansiyonu aktarın ve süpernatant atılır. </li><li> Yavaşça tam ortam içinde 1 ml makrofajlar yeniden süspanse edin. </li><li> 10 ul Tripan Mavisi solüsyonu ile makrofaj hücre süspansiyonu 10 ul karıştırın ve bir hemositometre kullanılarak makrofaj hücre sayısı ve canlılığı (tipik olarak&gt;% 95) belirler. </li><li> Hücre sayımına bağlı olarak (genel olarak yaklaşık 15-18 x 10 5 makrofajlar), arzu edilen tohumlama hücre yoğunluğu elde etmek için ek bir tam orta ekleyin. Not: 12 oyuklu plakanın her bir kuyucuğu için 1.5 ml ortam içinde 1 x 10 5 makrofajlar yakın bir Birleşik kültür üretecektir. </li><li> Tohumlamadan sonra, hücre tutunmasını sağlar,% 5 CO2 /% 95 hava ile 37 ° C hücre kültürü gece boyunca inkübatör içinde makrofajlar inkübe edin. Sonra, istenen deneyleri başlar. </li></ol>

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The authors have nothing to disclose.

tanımlı makrofaj türlerini üreten makrofaj biyoloji okuyan zaman araştırmacılar tarafından elde edilen çelişkili sonuçların bazılarını şöyle açıklamak mümkündür. çeşitli kültür koşulları ve farklılaşma faktörleri kullanımı primer insan makrofaj farklı makrofaj türlerine neden olabilir araştırmacı tarafından takdir olabilir bir gerçeği üretir. Örneğin, makrofajlar bazen serum tek başına insan serumu, FBS başına M-CSF, ya da M-CSF veya GM-CSF ihtiva eden FBS ile takviye edil...

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.

Tripan mavi 9 boyama ile saptandığı haliyle, taze veya dondurulmuş monosit canlılığı Şekil 1.% 95&#39;den daha fazla olan ve Şekil 2, alt ve üst büyütmelerde karşılaştırabilir sırasıyla makrofajlara taze ve dondurularak saklanmış (yani, donmuş) monosit farklılaşmasının ilerlemesi. Kriyoprezerve monositler yaymak ancak yuvarlak kalmaz monositler ayırt gösteren bir alt göstermektedir taze ka...

Watch this Scientific Journal Video about Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages at JoVE.com

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.

Makrofaj koloni stimüle edici faktör farklılaştırılmış insan monosit türevli makrofajlar Kültür

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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Immunology and Infection

29.1K Görüntüleme.  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.

Video: 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.

Bir<em&gt; In vitro</emİnsan Makrofaj Farklılaşma ve Polarizasyon heterojenliği Eğitim için&gt; Model

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

İmmünoloji ve Enfeksiyon

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.

İnsan Çift Gradient Santrifüj ile Monositlerinde ve teflon kaplı Hücre Kültürü Çantalar Makrofagların Onların Farklılaşma İzolasyonu

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.

Fare Kemik İliği itibaren Üretimi ve GM-CSF tanımlanması Türetilmiş Alveoler gibi makrofajlar ve dendritik hücrelerin

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.

Birincil İnsan Monositlerinin İzolasyon, Transfeksiyon ve Kültürü

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.

Diferansiye İnsan Monosit türevi Makrofajlarda Hücre Dışı Tuzak Salınımının İn Vitro Stimülasyon ve Görselleştirilmesi

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.

Monositlerin İnsan Kordon Kan Kök Hücresi (CB-SC)-Türetilmiş Ekzozomlar Ile Tedavi Sonrası Fenotipik Farklı Makrofajlara Farklılaşması

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.

makrofaj Farklılaşması ve Polarizasyonu, İnsan Monosit Benzeri THP-1 Lösemi Hücre Hattı Kullanarak M2 Benzeri Bir Fenotipe Dönüştür

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.

Farelerden Kemik İliği Kaynaklı Makrofajların İzolasyonu ve Kültürü

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://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).

İn Vitro Kültür ve Primer Monositlerin Makrofajlara Farklılaşması

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