Bu çalışma, Amerikan Kalp Derneği (Dr Beiyan Zhou için BGIA 7.850.037) tarafından desteklenmiştir.

1. Kemik İliği Hücreleri İzolasyon <ol><li> 6-8 haftalık farelerin femur ve tibia kemikleri izole, saç durulayın ve sonra kemik açık kesti. </li><li> Soğuk PBS +% 2 ısı inaktive fetal sığır serumu (FBS) (3-5 ml / fare) içine iliği dışarı atılması için bir 21G iğne ve 10 ml şırınga kullanın. </li><li> Hücreleri ayırmak için bir 21G iğne ile 4-6 kez iliği Min. </li><li> Hücre kümeleri, kemik, saç ve diğer hücre / doku kaldırmak için 70 mikron hücre süzgecinden hücreleri geçmektedir. </li><li> NH4CI çözeltisi (% 0.8 NH4CI çözeltisi, Kök Hücre Teknolojisi) 3 hacim eklemek ve kırmızı kan hücreleri çıkarmak için 10 dakika boyunca buz üzerinde inkübe edilir. </li><li> 4 ° C&#39;de 5 dakika boyunca 500 xg&#39;de hücreleri aşağı Spin </li><li> Soğuk PBS +% 2 FBS (20-50 ml, hücre miktarına bağlı olarak) içinde hücre pelletini. </li></ol> 2. Indüksiyon BMDM Oluşumu <ol><li> BMDM büyüme ortamı içinde izole edilmiş kemik iliği hücreleri (2x10 6 hücre / a süspansel). </li></ol> BMDM büyüme ortamı: Iscove Modifiye Dulbecco ortamı (IMDM) +% 10 FBS +% 15 süzüldü (0.2 um), L-929 hücresi (ATCC CCL-1) kültür yüzey (monosit-koloni uyarıcı faktör, M-CSF ihtiva eden) veya 10 ng / ml &#39;M -CSF verildi. Not: L-929 hücre yüzer M-CSF 18 içerir. Koşullandırılmış ortam, 5 etkin aktivitesi sağlamak için 5 x 10 L-929 hücreleri 6-7 gün boyunca T75 cm2 şişelerde tohumlanmıştır, kıvamlandırılmış ortam maddesi kullanımdan önce filtre bir 0.45 mikron ile toplanmış ve geçirilir. Orta alikotları 1-2 ay için hemen kullanılabilir ya da -80 ° C&#39;de saklanabilir. <ol start="2"><li> 6 veya 12 oyuklu doku kültürü plakaları (deney tasarımına bağlı olarak) (Corning Costar) tohum hücreleri. </li><li> 3. günde taze BMDM büyüme ortamı değiştirin. </li><li> 7. günde, olgun BMDM oluşumu akış sitometri analizi kullanılarak değerlendirilmiştirve fluorofor konjuge antikorlar CD11b ve F4/80 ifade hücreleri algılamak için. </li></ol> 3. BMDM Polarize Aktivasyon <ol><li> 7. günde taze ortam uyarılması değiştirin: M1 aktivasyonu için, IMDM 50 ng / ml, IFNy ile,% 10 FBS ve 100 ng / ml LPS ya da 100 ng / ml LPS içeren kullanımı, M2 aktivasyonu için,% 10 FBS içeren IMDM kullanmak 10 ng / ml IL-4 ve / veya 10 ng / ml IL-13. </li><li> Sıcak% 0.05 tripsin kullanarak çanak bunların ayrılması ile uyarılan BMDMs toplayın, PBS% 10 FBS içeren hücreler iki kez yıkanması takip etti. </li></ol> Not: farklılaşma sonra olgun makrofajlar,% 0.05 tripsin solüsyonu (0.48 mM EDTA, Invitrogen ihtiva eden) veya Ca ve Mg-içermeyen PBS veya Hank dengeli tamponu (HBSS) içerisinde 2-5 mM EDTA ayırmak ve yeniden süspanse etmek için kullanılabilir. Tripsin içeren sindirim orta kullanırken, hücrelerin sur kaybını önlemek için en az 10 dakika süreyle 37 ° C de tedavi ediliryüz aşırı sindirim nedeniyle proteinleri. <ol start="3"><li> CD11b F4/80, CD11c, CD206, CD69, CD80 ya da standart akış sitometrisi boyama işlemleri kullanarak, çeşitli zaman noktalarında CD86 dahil olmak üzere hücre yüzey antijenlerinin ekspresyonunu tespit etmek için antikorların kullanımı. </li><li> QRT-PCR kullanılarak, IL-1β, TNF-α ve IL-6 (M1 aktivasyonu) veya IL-10, IL-13, arginase1 ve PPARy (M2 aktivasyonu) dahil olmak üzere, aktive edilmiş makrofajlar M1 ve M2 karakteristik genlerin ekspresyonunu belirlemek. Batı blot analizi ile M1 veya M2 makrofaj aktivasyonu ile ilgili hücre sinyal yollarının aktivasyonu belirleyin. </li></ol>

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A., Derudas, B., Rigamonti, E., Dievart, R., Brozek, J., Haulon, S., Zawadzki, C., Jude, B., Torpier, G., Marx, N., Staels, B., Chinetti-Gbaguidi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pparg+activation+primes+human+monocytes+into+alternative+m2+macrophages+with+anti-inflammatory+properties.">Pparg activation primes human monocytes into alternative m2 macrophages with anti-inflammatory properties.</a> Cell Metabolism. 6, 137-143 (2007).</li><li>Bronte, V., Zanovello, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+immune+responses+by+l-arginine+metabolism.">Regulation of immune responses by l-arginine metabolism.</a> Nat. Rev. Immunol. 5, 641-654 (2005).</li><li>Zhuang, G., Meng, C., Guo, X., Cheruku, P. S., Shi, L., Xu, H., Li, H., Wang, G., Evans, A. 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Çıkar çatışması ilan etti.

Burada kemik iliği projenitör hücrelerinden elde edilen makrofajların aktivasyonu uyarmak için basit ve in vitro olarak kolaylıkla adapte prosedür sunulmuştur. Bu prosedür, makrofaj polarizasyon sorumlu mekanizmalarının incelenmesi için de kullanılabilir. Olgun makrofajlar saflığı bu protokolü ortalama 95 ila 99% kullanılarak elde ve ek saflaştırma işlemleri gereklidir. Makrofaj polarizasyon, ektopik ifade veya gen spesifik demonte bağlamında özel ilgi genlerin işlevine araştırmak 7. ...

Klasik inflamatuar yanıtları, dokuların sızmak makrofajlar farklı genellikle konak doku fizyolojik fonksiyonları 1-8 düzenlenmesinde önemli bir rol oynar polarize etkinleştirme durumunu görüntüler. Stimülasyon üzerine, makrofaj aktivasyonu klasik (M1) ve alternatif (M2) aktivasyon 2, 4, 9 içine sıralanabilir. M1, makrofaj aktivasyonu, TNF-α ve benzeri gibi enflamatuar sitokinlerin üretimine yol açan bir Toll benzeri reseptör (TLR), ve nükleer faktör kappa B (NFKB) / c-Jun N-terminal kinaz 1 (JNK1) aktivasyonu, bağlıdır, IL- iNOS 1β ve aktivasyon böyle nitrit oksit (NO), 10, 11 gibi reaktif oksijen türlerinin artan üretimi ile sonuçlanır. Bunun aksine, M2 makrofaj aktivasyonu işe PPARy, PPARd, veya IL-4-STAT6 yolları, alternatif manoz reseptörü CD206 upregülasyonunu ile ilişkili, bir anti-enflamatuar (M2) aktivasyonu ve arginaz 1 (Arg1) 6, 12, önde gelen - 14 &lt;/&gt; Sup. Kemik iliği kökenli makrofajlar (BMDM) aktive makrofajlar 15 kutuplaşma kontrol mekanizmaları anlamak için in vitro model ideal mevcut. M2 makrofaj polarizasyon, IL-4 ve / veya IL-13 ile indüklenen edilebilir ise Özellikle, M1, makrofajların aktivasyonu, lipopolisakkarit (LPS) ile uyarılması sonucu meydana edilebilir. Olgun kemik iliği kökenli makrofajlar ve aktive makrofajlar CD11b, F4/80, CD11c, CD206, CD69, CD80 ve CD86 9, 16, 17 de dahil olmak üzere yüzey antijenleri, ifadesi için flow sitometri analizi ile tespit edilebilir. Buna ek olarak, sitokin üretimi ve makrofaj kutuplaşma ile bağlantılı hücre sinyalizasyon yolları değişiklikler kantitatif RT-PCR ve Batı sırasıyla blot ile ölçülebilir. Özetle, fare kemik iliği kökenli makrofajlar in vitro makrofaj kutuplaşma incelemek için ilgili model olarak hizmet edebilir.

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>IMDM</td>
 <td>Thermo Scientific</td>
 <td>SH30259.01</td>
 <td></td>
 </tr>
 <tr>
 <td>Fetal bovine serum</td>
 <td>Invitrogen</td>
 <td>10438-026</td>
 <td></td>
 </tr>
 <tr>
 <td>Murine GM-CSF</td>
 <td>PeproTech</td>
 <td> 315-03</td>
 <td></td>
 </tr>
 <tr>
 <td>NH4Cl</td>
 <td>StemCell Technologies</td>
 <td>7850</td>
 <td></td>
 </tr>
 <tr>
 <td>L-929</td>
 <td>ATCC</td>
 <td>CCL-1</td>
 <td></td>
 </tr>
 <tr>
 <td>70 &mu;m cell strainer</td>
 <td>BD Biosciences</td>
 <td>352350</td>
 <td></td>
 </tr>
 <tr>
 <td>10 x PBS</td>
 <td>Thermo Scientific</td>
 <td>AP-9009-10</td>
 <td></td>
 </tr>
 <tr>
 <td>Anti-mouse CD11b-APC</td>
 <td>eBioscience</td>
 <td>17-0112-81</td>
 <td></td>
 </tr>
 <tr>
 <td>Anti-mouse F4/80-FITC</td>
 <td>eBioscience</td>
 <td>11-4801-81</td>
 <td></td>
 </tr>
 <tr>
 <td>Anti-mouse CD69-PE</td>
 <td>eBioscience</td>
 <td>12-0691-81</td>
 <td></td>
 </tr>
 <tr>
 <td>Anti-mouse CD86-PE</td>
 <td>eBioscience</td>
 <td>12-0862-81</td>
 <td></td>
 </tr>
 <tr>
 <td>Propidium Iodine</td>
 <td>Invitrogen</td>
 <td>P3566</td>
 <td></td>
 </tr>
 </tbody>
</table>

investigation of macrophage polarization using bone marrow derived macrophages

Makalede, makrofaj polarizasyon araştırmak için in vitro model, kolayca kolayca uyarlanabilir açıklanmaktadır. GM-CSF/M-CSF varlığında, kemik iliği hematopoetik kök / progenitör hücrelerin M1 veya M2 uyarımı ile devam eden, monositik farklılaşma içine yönlendirilir. Etkinleştirme durumunu hücre yüzey antijenleri, gen ve hücre sinyal yolları değişiklikleri ile takip edilebilir.

BMDM nesil prosedürün şematik bir açıklama (Şekil 1) sunulmuştur. Onlar CD11b + F4/80 + hücreler 95% 99 (Şekil 2) temsil zaman olgun makrofaj yüksek saflıkta 7. günde görülebilir. Polarize makrofajlar CD11b, F4/80 karşı antikor kullanılarak incelenebilir, CD11c ve CD206 akım sitometri analizi ile izledi. Şekil 3 &#39;de gösterildiği gibi, M1 makrofajlar gibi tespit edilir CD11b + F4/80 + CD11c + CD206-hücrelerinin (Q2), M2 makrofajlar CD11b oysa + ...

Watch this Scientific Journal Video about Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages at JoVE.com

Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

Bu makale, kolayca kolay uyarlanabilir açıklar<em&gt; In vitro</emMakrofaj polarizasyon araştırmak için&gt; modeli. GM-CSF/M-CSF varlığında, kemik iliği hematopoetik kök / progenitör hücrelerin M1 veya M2 uyarımı ile devam eden, monositik farklılaşma içine yönlendirilir. Etkinleştirme durumunu hücre yüzey antijenleri, gen ve hücre sinyal yolları değişiklikleri ile takip edilebilir.

Kemik İliği Türetilmiş makrofajlar kullanarak Makrofaj Polarizasyon İncelenmesi

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

investigation-macrophage-polarization-using-bone-marrow-derived

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JoVE Journal

Immunology and Infection

65.6K Görüntüleme.  Texas A&M University. Bu makale, kolayca kolay uyarlanabilir açıklar In vitro modeli. GM-CSF/M-CSF varlığında, kemik iliği hematopoetik kök / progenitör hücrelerin M1 veya M2 uyarımı ile devam eden, monositik farklılaşma içine yönlendirilir. Etkinleştirme durumunu hücre yüzey antijenleri, gen ve hücre sinyal yolları değişiklikleri ile takip edilebilir.

Video: Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of warm-blooded animals. During infection, T. gondii disseminates as a fast replicating form called the tachyzoite. Tachyzoites convert into a slow-growing encysted form called the bradyzoite by a signaling process that is not well characterized. Within animals, bradyzoite cysts are found in the central nervous system and muscle tissue and represent the chronic stage of infection. Conversion to bradyzoites can be simulated in tissue culture by CO2 starvation, using medium with high a pH, or the addition of interferon gamma (IFN&gamma;). Bradyzoites are characterized by the presence of a cyst wall, to which the lectin Dolichos biflorus agglutinin (DBA) binds. Fluorescently labeled DBA is used to visualize the cyst wall in parasites grown in human foreskin fibroblasts (HFFs) that have been exposed to low CO2 and high pH medium. Similarly, parasites residing in murine bone marrow-derived macrophages (BMMs) display a cyst wall detectable by DBA after the BMMs are activated with IFN&gamma; and lipopolysaccharide (LPS). This protocol will demonstrate how to induce conversion of T. gondii to bradyzoites using a high pH growth medium with low CO2 and activation of BMMs. Host cells will be cultured on coverslips, infected with tachyzoites and either activated with addition of IFN&gamma; and LPS (BMMs) or exposed to a high pH growth medium (HFFs) for three days. Upon completion of infections, host cells will be fixed, permeabilized, and blocked. Cyst walls will be visualized using rhodamine DBA with fluorescence microscopy.

Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

Toxoplasma gondii converts to a cyst form in response to environmental stresses, which can be mimicked in tissue culture models. This video demonstrates techniques to examine cyst wall formation by activating bone marrow-derived macrophages or changing growth medium pH in fibroblast cells.

Aktif Kemik İliği türetilen Makrofajlar ve Bradyzoite Koşullar Toxoplasma gondii Kist Duvar Oluşumu

Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of warm-blooded animals. During infection, T. gondii ...

İmmünoloji ve Enfeksiyon

Bone Marrow-derived Macrophage Production

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because of their powerful microbicidal activities. Macrophages are widely distributed throughout the body and are present in the lymphoid organs, liver, lungs, gastrointestinal tract, central nervous system, bone, and skin. Because of their repartition, they participate in a wide range of physiological and pathological processes. Macrophages are highly versatile cells that are able to recognize microenvironmental alterations and to maintain tissue homeostasis. Numerous pathogens have evolved mechanisms to use macrophages as Trojan horses to survive, replicate in, and infect both humans and animals and to propagate throughout the body. The recent explosion of interest in evolutionary, genetic, and biochemical aspects of host-pathogen interactions has renewed scientific attention regarding macrophages. Here, we describe a procedure to isolate and cultivate macrophages from murine bone marrow that will provide large numbers of macrophages for studying host-pathogen interactions as well as other processes.

Macrophages have long been recognized as a critical component of the innate and adaptive immune responses. The recent explosion of knowledge pertaining to evolutionary, genetic, and biochemical aspects of the interaction between macrophages and microbes has renewed scientific attention to macrophages. This article describes a method to differentiate macrophages from mouse bone marrow.

Kemik İliği Makrofaj üretim kaynaklı

Macrophages are critical components of the innate and adaptive immune responses, and they are the first line of defense against foreign invaders because ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

İzolasyon ve Kemirgen Kemik İliği intravenöz enjeksiyon Türetilmiş Monositler

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Metabolic Characterization of Polarized M1 and M2 Bone Marrow-derived Macrophages Using Real-time Extracellular Flux Analysis

Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or M(LPS) macrophages are pro-inflammatory, IL-4 induces alternative macrophage activation and these so-called M2 or M(IL-4) support resolution of inflammation and wound healing. Recent evidence shows the crucial role of metabolic reprogramming in the regulation of M1 and M2 macrophage polarization. 
In this manuscript, an extracellular flux analyzer is applied to assess the metabolic characteristics of naive, M1 and M2 polarized mouse bone marrow-derived macrophages. This instrument uses pH and oxygen sensors to measure the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), which can be related to glycolytic and mitochondrial oxidative metabolism. As such, both glycolysis and mitochondrial oxidative metabolism can be measured in real-time in one single assay.
Using this technique, we demonstrate here that inflammatory M1 macrophages display enhanced glycolytic metabolism and reduced mitochondrial activity. Conversely, anti-inflammatory M2 macrophages show high mitochondrial oxidative phosphorylation (OXPHOS) and are characterized by an enhanced spare respiratory capacity (SRC). 
The presented functional assay serves as a framework to investigate how particular cytokines, pharmacological compounds, gene knock outs or other interventions affect the macrophage&#8217;s metabolic phenotype and inflammatory status.

Metabolic reprogramming is a characteristic and prerequisite for M1 and M2 macrophage polarization. This manuscript describes an assay for the measurement of fundamental parameters of glycolysis and mitochondrial function in mouse bone marrow-derived macrophages. This tool can be applied to investigate how particular factors affect the macrophage&#8217;s metabolism and phenotype.

Gerçek zamanlı Ekstrasellüler Flux Analizi Polarize M1 ve M2 Kemik İliği-türevli Makrofagların Metabolik Karakterizasyonu

Specific metabolic pathways are increasingly being recognized as critical hallmarks of macrophage subsets. While LPS-induced classically activated M1 or ...

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. ...

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.

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 ...

Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages are equally important in turning off inflammatory responses. We have shown that macrophages stimulated with intravenous immunoglobulin (IVIg) can produce high amounts of the anti-inflammatory cytokine, interleukin 10 (IL-10), and low levels of pro-inflammatory cytokines in response to bacterial lipopolysaccharides (LPS). IVIg is a polyvalent antibody, primarily immunoglobulin Gs (IgGs), pooled from the plasma of more than 1,000 blood donors. It is used to supplement antibodies in patients with immune deficiencies or to suppress immune responses in patients with autoimmune or inflammatory conditions. Infliximab, a therapeutic anti-tumor necrosis factor alpha (TNF&#945;) antibody, has also been shown to activate macrophages to produce IL-10 in response to inflammatory stimuli. IVIg and other antibody-based biologics can be tested to determine their effects on macrophage activation. This paper describes methods for derivation, stimulation, and assessment of murine bone marrow macrophages activated by antibodies in vitro and murine peritoneal macrophages activated with antibodies in vivo. Finally, we demonstrate the use of western blotting to determine the contribution of specific cell signaling pathways to anti-inflammatory macrophage activity. These protocols can be used with genetically modified mice, to determine the effect of a specific protein(s) on anti-inflammatory macrophage activation. These techniques can also be used to assess whether specific biologics may act by changing macrophages to an IL-10-producing anti-inflammatory activation state that reduces inflammatory responses in vivo. This can provide information on the role of macrophage activation in the efficacy of biologics during disease models in mice, and provide insight into a potential new mechanism of action in people. Conversely, this may caution against the use of specific antibody-based biologics to treat infectious disease, particularly if macrophages play an important role in host defense against that infection.

Antibody-based drugs have revolutionized treatment for inflammatory diseases. In addition to having direct effects on specific targets, antibodies can activate macrophages to become anti-inflammatory. This protocol describes how anti-inflammatory macrophage activation can be assessed in vitro, using mouse bone marrow macrophages, and in vivo, using peritoneal macrophages.

Fare kemik iliği ve Periton makrofaj harekete geçirmek antikor bazlı ilaçlara etkileri değerlendirilmesi

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages ...

Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote inflammatory and autoinflammatory diseases. Inflammasomes are activated by cytosolic pattern recognition receptors, including members of the NOD-like receptor (NLR) family. These receptors oligomerize upon the detection of microbial or damage-associated stimuli. Subsequent recruitment of the adaptor protein ASC forms a microscopically visible inflammasome complex, which activates caspase-1 through proximity-induced auto-activation. Following the activation, caspase-1 cleaves pro-IL-1&#946; and pro-IL-18, leading to the activation and secretion of these pro-inflammatory cytokines. Caspase-1 also mediates the inflammatory form of cell death termed pyroptosis, which features the loss of membrane integrity and cell lysis. Caspase-1 cleaves gasdermin D, releasing the N-terminal fragment which forms plasma membrane pores, leading to osmotic lysis.
In vitro, the activation of caspase-1 can be determined by labeling bone marrow-derived macrophages with the caspase-1 activity probe FAM-YVAD-FMK and by labeling the cells with antibodies against the adaptor protein ASC. This technique allows the identification of inflammasome formation and caspase-1 activation in individual cells using fluorescence microscopy. Pyroptotic cell death can be detected by measuring the release of cytosolic lactate dehydrogenase into the medium. This procedure is simple, cost effective and performed in a 96-well plate format, allowing adaptation for screening. In this manuscript, we show that activation of the NLRP3 inflammasome by nigericin leads to the co-localization of the adaptor protein ASC and active caspase-1, leading to pyroptosis.

We describe the detection of NLRP3 inflammasome activation on cellular basis using fluorescence microscopy and staining for active caspase-1 and the adaptor, ASC. A lactate dehydrogenase release assay is presented to detect pyroptotic lysis on a population basis. These techniques can be adapted to study many aspects of inflammasome biology.

Inflammasome harekete geçirmek ve Pyroptotic hücre ölümü fare kemik iliği türevi makrofajlar içinde tespiti

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote ...

Isolation and In vitro Culture of Bone Marrow-Derived Macrophages for the Study of NO-Redox Biology

Macrophages are derived from hematopoietic progenitor cells throughout the body, are central to inflammatory processes, and participate in innate and adaptive immune responses. In vitro study of macrophages can be undertaken by ex vivo culture from the peritoneum or through differentiation of myeloid bone marrow progenitor cells to form bone marrow-derived macrophages (BMDMs). A common approach to macrophage differentiation from precursors involves the use of conditioned media from L929 cells (LCM). This media is easy to self-produce but suffers from batch variability, and its constituents are undefined. Similarly, Foetal Bovine Serum (FBS) is used to support growth but contains a vast mixture of undefined molecules that may vary between batches. These methods are not adequate for the study of nitric oxide biology and redox mechanisms as they both contain substantial amounts of small molecules that either interfere with redox mechanisms or supplement levels of cofactors, such as tetrahydrobiopterin (BH4), required for the production of NO from inducible nitric oxide synthase (iNOS). In this report, we present an optimized protocol allowing for control of the NO-redox environment by reducing the levels of exogenous biopterin while maintaining conditions suitable for cell growth and differentiation. Tight control of culture media composition helps ensure experimental reproducibility and facilitates accurate interpretation of results. In this protocol, BMDMs were obtained from a GTP cyclohydrolase (GCH)- deficient mouse model. Culture of BMDMs was performed with media containing either (i) conditioned LCM, or (ii) recombinant M-CSF and GM-CSF to produce minimal artifacts while obtaining BH4 and NO-deficient culture conditions - thus allowing for the reproducible study of NO-redox biology and immunometabolism in vitro.

Isolation and In vitro Culture of Bone Marrow-Derived Macrophages for the Study of NO-Redox Biology

This protocol has been established to culture tetrahydrobiopterin (BH4)- and inducible nitric oxide synthase (iNOS)-deficient primary murine macrophages to study NO-redox biology. The study focuses on reducing potential contamination of BH4 and other artifacts found in traditional isolation and culture methods which may confound experimental outcomes and interpretation of results.

NO-Redoks Biyolojisi Çalışması için Kemik İliği Kaynaklı Makrofajların İzolasyonu ve In vitro Kültürü

Macrophages are derived from hematopoietic progenitor cells throughout the body, are central to inflammatory processes, and participate in innate and ...

Economical and Efficient Protocol for Isolating and Culturing Bone Marrow-derived Dendritic Cells from Mice

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method for isolating DCs primarily involves inducing bone marrow (BM) differentiation into DCs by injecting large doses (&#62;10 ng/mL) of granulocyte-macrophage colony-stimulating factor/interleukin-4 (GM-CSF/IL-4), making the procedure complex and expensive. In this protocol, using all BM cells cultured in 10 ng/mL GM-CSF/IL-4 medium, after 3-4 half-culture exchanges, up to 2.7 x 107 CD11c+ cells&#160;(DCs) per mouse (two femurs) were harvested with a purity of 80%-95%. After 10 days in culture, the expression of CD11c, CD80, and MHC II increased, whereas the number of cells decreased. The number of cells peaked after 7 days of culture. Moreover, this method only took 10 min to harvest all bone marrow cells, and a high number of DCs were obtained after 1 week of culture.

Here, we present an economical and efficient method to isolate and generate high-purity bone marrow-derived dendritic cells from mice after 7 days of culture with 10 ng/mL GM-CSF/IL-4.

Kemik İliği Kaynaklı Dendritik Hücrelerin Farelerden İzole Edilmesi ve Kültürlenmesi için Ekonomik ve Verimli Protokol

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method ...