Biz, değerli katkılarından dolayı K. Nakagaki Dr H. Ishii, Dr. K. Suzuki, A. Yamagata, K. Oofusa müteşekkiriz.

1. SGMRα üretimi ve saflaştırılması <ol><li> Polimeraz zincir reaksiyonu (PCR) ile insan plasenta cDNA kütüphanesi sGMRα, cDNA artırın. </li><li> His-tag 3 &#39;başka bir PCR PCR Amplikon sonuna kadar 50 baz bakülovirüs polyhedrin sonu 5&#39;-UTR dizisi ve 27 baz kodlama RGS ekleyin. </li><li> Çoğaltılan PCR ürününün bir plazmid pMSG1.1MG içine yerleştirin. </li><li> Ipekböceği PND-w1 suşu yumurta içine pHA3PIG yardımcı vektörü ile yapı psGMR/M1.1MG enjekte edilir. </li><li> 25 kelebekler Arka yumurtadan G0 larvaları ° C SGMRα geni taşıyan transgenik böceklerinin elde etmek için gözlerini MGFP ifade için kardeşler arasında ya da PND-w1 çiftleşme elde Ekran G1 embriyolar. </li><li> 500mm NaCl içeren fosfat tamponu ile transgenik böceklerinin koza rekombinant sGMRα ayıklayın ve 4 karıştırın ° C 24 saat için. </li><li> 15dk 20000 g örnek bir çökeltileri kaldırmak için santrifüjleyin. </li><li> Nikel afiniteli bir sütun supernatant uygulayın. </li><li> 10mM imidazol, 500mm NaCl ve 20mm Sodyum fosfat, pH6.3 ve 10mm den 250mm imidazol doğrusal bir degrade Zehir sütun yıkayın. (PH7.4) karşı saflaştırılmış sGMRα ve dialyze içeren kesirler Havuzu fosfat tamponlu salin solüsyonu (PBS). </li></ol> 2. Rekombinant GM-CSF, biyotinilasyon <ol><li> PBS karşı diyaliz sorbitol hazırlık çıkarın. </li><li> 4 20mm soğuk sodyum meta-periyodat çözüm ° C buz üzerinde çözüm 1ml ekleyin. </li><li> Için örnek karanlıkta buz üzerinde 30 dk inkübe edin. </li><li> 15mm son bir konsantrasyonda çözüm gliserol ekleyin. </li><li> 100 mM sodyum asetat tamponu (pH5.5) karşı çözüm Dialyze </li><li> 5mM son bir konsantrasyonda çözüm biotin hydrazide ekleyin ve oda sıcaklığında 2 saat için çözüm sürekli ajitasyon. </li><li> PBS, pH7.4 karşı diyaliz olmayan tepki malzeme çıkarın. </li></ol> 3. Bir hücre GM-CSF reseptör-ligand bağlayıcı test <ol><li> 4 gece anti-polyHistidine monoklonal antikor 50μl Coat mikrotiter plate ° C </li><li> PBST, 500μl beş kez yıkayın. </li><li> 4 3h engelleyici bir çözüm ve inkübe ° C 100μl ekle </li><li> PBST, 500μl beş kez yıkayın. </li><li> Engelleme çözüm sGMRα, 50μl ekleyin ve 4 gece inkübe ° C </li><li> PBST, 500μl beş kez yıkayın. </li><li> GM-CSF otoantikor ve biotinlenmiş GM-CSF 25μl içeren örnekleri 25μl ekle. 1s için 4 ° C&#39;de sGMRα-GM-CSF kompleks oluşturacak şekilde. </li><li> PBST, 500μl beş kez yıkayın. </li><li> 1 saat için 0.4 streptavidin alkalen fosfataz 50μl 4 ° C biotinlenmiş GM-CSF sınır sGMRα algılamak için. </li><li> PBST, 500μl beş kez yıkayın. </li><li> Çözüm için bir kemilüminesan substrat 50μl ve 1s oda sıcaklığında inkübe. </li><li> Kemilüminesans plaka okuyucu kullanarak, kemilüminesans faaliyeti algılar. </li></ol> 4. Temsilcisi Sonuçlar: GM-CSF reseptör sinyal iletim yolağı ilk adım hücre yüzeyinde GM-CSF, GM-CSF reseptör alfa bağlayıcıdır. GM-CSF otoantikor özellikle GM-CSF bağlar ve in vitro 16,17 reseptör bağlanma blok Çünkü otoantikorlar GM-CSF doğrudan bağlanarak bu ilk tepkisi inhibe ettiği hipotezini. (Ref. 9-15), daha önce açıklandığı gibi yüksek saflıkta ile rekombinant sGMRα büyük miktarda elde etmek için transgenik ipekböceği teknolojisi uygulanır. Ipekböceği edilen rekombinant sGMRα düşürücü olmayan koşullar altında SDS-PAGE yüklü iken, sadece monomerik formu azaltarak koşulları altında tespit iken sGMRα (Şekil 1B), her iki monomerik (45 kDa) ve dimerik (90 kDa) formları gösterdi monomerlerin ve disülfit bağlantılı dimmer 18 rekombinant sGMRα bir karışımı olduğunu gösterir. Kullanarak, hücre sistemi (Şekil 2A), GM-CSF otoantikorlar (Şekil 2B ve Şekil 2C) tarafından sGMRα, GM-CSF bağlama inhibisyonu değerlendirildi. Bu yöntemler URANO ve arkadaşları tarafından 19 referans tarif edildi. Büyüme inhibisyonu GM-CSF otoantikor (Şekil 3A) 19 çeşitli konsantrasyonlarda bağlayıcı inhibisyonu (r = 0,988, p = 0.002) ile yakından ilişkili oldu. Benzer şekilde, bağlayıcı inhibisyonu önemli ölçüde büyüme inhibisyonu ile korele bulunmuştur. Bağlayıcı inhibisyonu GM-CSF otoantikorlar (Şekil 3B) 19 doza bağımlı bir şekilde artmıştır . Bu iki parametre farklı hastadan alınan serum IgG fraksiyonları birbirleriyle korele idi (r = 0.589 p = 0.006, Şekil 3C). Ne bağlayıcı inhibisyonu ne de büyüme inhibisyonu Kd değerleri ile ilişkili ve bu nedenle, her iki parametre afinitesi 19 etkilenmemiştir . Sonuç olarak tekrarlanabilirlik bağlayıcı ve g arasında veriüç bağımsız üç farklı örnekleri üzerinde deneyler yoluyla elde rowth inhibisyonu değerlendirildi. Varyasyon katsayısı hücre sistemi biyoassay (Tablo 1) daha mükemmel olduğunu belirtti. <img src="/files/ftp_upload/2742/2742fig1.jpg" alt="Şekil 1" /> Şekil 1 transgenik böceklerinin kullanarak sGMRα üretimi için prosedürün Akış şeması. A) dönüşüm vektörleri 9-11 Yapıları. B) SDS-PAGE ve Coomassie azaltılması ve azaltıcı olmayan koşullar altında saflaştırılmış sGMRα Brillant Blue boyama. sGMRα, çözünür GM-CSF reseptör alfa; MGFP, canavar yeşil flüoresan protein BmNPVpol5&#39;-UTR, Bombyx mori nükleer polyhedrosis virüs polyhedrin 5&#39;-çevrilmemiş bölge sırası; SV40 Polya, SV40 Polya sinyal dizisi, P 3xP3, 3xP3 organizatörü, P ser1 ser1 organizatörü; fibL Polya, fibroin L-zincir Polya sinyal dizisi; hr3, Bombyx mori nükleer polyhedrosis virüs hr3 arttırıcı. <img src="/files/ftp_upload/2742/2742fig2.jpg" alt="Şekil 2" /> Şekil 2 hücre test sistemi şeması. A) İpek böceği tarafından üretilen sGMRα kullanarak rekabet bağlayıcı assay. B) Etkisi nötralize ve non-nötralize edici antikorlar hücre sistemi ile bağlayıcı inhibisyonu. C) nötralize edici antikorlar ve non-nötralize edici antikorlar çeşitli konsantrasyonlarda arasında bağlayıcı inhibisyonu fark. GM-CSF, granülosit makrofaj koloni-uyarıcı faktör; sGMRα, çözünür GM-CSF reseptör alfa; Onun, RGS-His-tag; AP, alkalin fosfataz. <img src="/files/ftp_upload/2742/2742fig3.jpg" alt="Şekil 3" /> Şekil 3 sGMRα GM-CSF, GM-CSF poliklonal antikorlar veya otoimmün PAP olan hastalarda serum IgG kesirler etkisi bağlayıcı inhibisyonu . A) GM-CSF otoantikor bağlayıcı inhibisyonu ve büyüme inhibisyonu arasındaki ilişki. GM-CSF otoantikor çeşitli konsantrasyonlarda B) Cilt inhibisyonu. C), yüzde bağlayıcı inhibisyonu serum IgG kesirler 19 ve yüzde büyüme inhibisyonu için IC 50 arasında ilişki . IC 50,% 50 inhibitör konsantrasyon; GM-CSF, granülosit-makrofaj koloni-uyarıcı faktör. <table border="1"><tbody><tr><td> Örnek </td><td> A </td><td> B </td><td> C </td></tr><tr><td colspan="4"> Inter-assay </td></tr><tr><td> # Tespitler </td><td> 3 </td><td> 3 </td><td> 3 </td></tr><tr><td> Ortalama değer (% bağlayıcı inhibisyonu) </td><td> 61,6 </td><td> 63,8 </td><td> 69,7 </td></tr><tr><td> Ortalama değer (% büyüme inhibisyonu) </td><td> 21,0 </td><td> 73,4 </td><td> 82,8 </td></tr><tr><td> Varyasyon katsayısı (%) (% bağlayıcı inhibisyonu) </td><td> 6,0 </td><td> 5,6 </td><td> 8,3 </td></tr><tr><td> Varyasyon katsayısı (%) (% büyüme inhibisyonu) </td><td> 64,4 </td><td> 18,3 </td><td> 10,4 </td></tr></tbody></table> Her iki Testler 5 ng / ml, GM-CSF ve GM-CSF otoantikor eşit bir konsantrasyon yapıldı. Tablo 1 üç bağımsız deneyler yoluyla elde edilen yüzde bağlama ve büyüme inhibisyon arasındaki varyasyon katsayısı karşılaştırılması .

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Hücre ücretsiz test GM-CSF otoantikorların mükemmel tekrarlanabilirlik ve hızla nötralize kapasitesi tahmin edilmektedir. GM-CSF otoantikorlar veya hastanın serum IgG kesirler bağlayıcı inhibisyon testi ile değerlendirildi. Veri hücre testinin bağlayıcı inhibisyonu ve TF-1 hücreleri, sırasıyla kullanarak biyoassay büyüme inhibisyonu arasında bir korelasyon gösterdi. Biyoassay yaygın olarak kullanılan, ancak farklı tesis ve biz bu yeni sistem kullanarak önleyebilirsiniz farklı zaman noktaları ...

a cell free assay system estimating the neutralizing capacity of gm-csf antibody using recombinant soluble gm-csf receptor

ZEMİN: Daha önce, biz bu nötralize kapasitesi gösterdi ama GM-CSF otoantikor konsantrasyonu otoimmün pulmoner alveoler proteinozis (PAP) 1-3 olan hastalarda hastalığın şiddeti ile korelasyon. Akciğer GM-CSF biyoaktivite kaldırılması 4,5 otoimmün PAP için olası nedeni olarak, PAP ile her hastada hastalık şiddeti değerlendirmek için, GM-CSF otoantikorların nötralize kapasitesini ölçmek için umut verici . Şimdiye kadar, GM-CSF otoantikorların nötralize kapasitesi, insan kemik iliği hücreleri ya da GM-CSF 6-8 ile uyarılan TF-1 hücreleri büyüme inhibisyonu değerlendirerek değerlendirilmiştir. Biyoassay sisteminde, ancak sabit bir durum hücrelerin korunmasında teknik zorluklar nedeniyle farklı laboratuvarlarda verileri karşılaştırmak için güvenilir verilerin yanı sıra, elde etmek için çoğu zaman sorunlu. AMAÇ: GM-CSF hücre reseptör-bağlayıcı-assay kullanarak hücre yüzeyinde GM-CSF reseptör bağlayıcı taklit etmek için. YÖNTEM: Transgenik ipekböceği teknolojisi, yüksek saflıkta 9-13 rekombinant çözünür, GM-CSF reseptör alfa (sGMRα) için büyük miktarda elde etmek için uygulandı. Rekombinant sGMRα ipek proteinleri erimiş olmadan ipek ipler hidrofilik serizin katmanlarda yer aldığını ve böylece, biz nötr sulu çözeltileri 14,15 ile kolayca iyi saflık koza elde edebilirsiniz. Neyse ki, GM-CSF ile bağlama için kritik olan oligosakkarit yapılar, diğer böcekler ya da mayalar tarafından üretilen dışında insan sGMRα yapılarını daha benzer. Bulgular: sGMRα kullanarak hücre test sistemi, yüksek plastisite ve güvenilirliği ile veri vermiştir. SGMRα için bağlayıcı GM-CSF sGMRα kullanarak yeni hücre ücretsiz test sistemi nötralize aktivitesinin ölçümü için daha yararlı olduğunu belirterek, benzer bir şekilde TF-1 hücreleri kullanarak biyoassay poliklonal GM-CSF otoantikor tarafından inhibe doza bağımlı TF-1 hücre veya insan kemik iliği hücreleri kullanarak biyoassay sistemine göre GM-CSF otoantikorların. Sonuç: GM-CSF otoantikor nötralize kapasitesi niceleme bir hücre ücretsiz tahlil kurdu.

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A Cell Free Assay System Estimating the Neutralizing Capacity of GM-CSF Antibody using Recombinant Soluble GM-CSF Receptor

Hücre reseptör bağlayıcı tahlil reseptörlerine granülosit-makrofaj koloni uyarıcı faktör (GM-CSF) bağlama tahmin etmek için tasarlanmıştır. Bu mükemmel tekrarlanabilirlik ile GM-CSF otoantikor çözünen GM-CSF reseptör alfa bağlayıcı biotinlenmiş GM-CSF yarışmalı inhibisyonu değerlendirmek için bize sağlar.

Rekombinant Çözünür GM-CSF Reseptör kullanarak GM-CSF Antikor Nötralizasyon Kapasitesi tahmin Hücre Testi Sistemi

BACKGROUNDS: Previously, we demonstrated that neutralizing capacity but not the concentration of GM-CSF autoantibody was correlated with the disease ...

a-cell-free-assay-system-estimating-neutralizing-capacity-gm-csf

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JoVE Journal

Immunology and Infection

12.6K Görüntüleme.  Niigata University Medical and Dental Hospital. Hücre reseptör bağlayıcı tahlil reseptörlerine granülosit-makrofaj koloni uyarıcı faktör (GM-CSF) bağlama tahmin etmek için tasarlanmıştır. Bu mükemmel tekrarlanabilirlik ile GM-CSF otoantikor çözünen GM-CSF reseptör alfa bağlayıcı biotinlenmiş GM-CSF yarışmalı inhibisyonu değerlendirmek için bize sağlar.

Video: A Cell Free Assay System Estimating the Neutralizing Capacity of GM-CSF Antibody using Recombinant Soluble GM-CSF Receptor

Bioenergetics and the Oxidative Burst: Protocols for the Isolation and Evaluation of Human Leukocytes and Platelets

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and neurodegenerative diseases but assessing changes in bioenergetic function in patients is challenging.&#160;Although diseases such as diabetes or atherosclerosis present clinically with specific organ impairment, the systemic components of the pathology, such as hyperglycemia or inflammation, can alter bioenergetic function in circulating leukocytes or platelets. This concept has been recognized for some time but its widespread application has been constrained by the large number of primary cells needed for bioenergetic analysis.&#160;This technical limitation has been overcome by combining the specificity of the magnetic bead isolation techniques, cell adhesion techniques, which allow cells to be attached without activation to microplates, and the sensitivity of new technologies designed for high throughput microplate respirometry.&#160;An example of this equipment is the extracellular flux analyzer. Such instrumentation typically uses oxygen and pH sensitive probes to measure rates of change in these parameters in adherent cells, which can then be related to metabolism. Here we detail the methods for the isolation and plating of monocytes, lymphocytes, neutrophils and platelets, without activation, from human blood and the analysis of mitochondrial bioenergetic function in these cells. In addition, we demonstrate how the oxidative burst in monocytes and neutrophils can also be measured in the same samples. Since these methods use only 8-20 ml human blood they have potential for monitoring reactive oxygen species generation and bioenergetics in a clinical setting.

Blood leukocytes and platelets can be used as a marker of overall bioenergetic health of an individual and so have the potential to monitor pathological processes and the impact of treatments. Here&#160;we describe a method to isolate and measure mitochondrial function and the oxidative burst in these cells.

İnsan lökosit ve trombosit İzolasyonu ve Değerlendirme Protokolleri: Biyoenerji ve Oksidatif Burst

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and ...

İmmünoloji ve Enfeksiyon

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.

A method is described with visual accompaniment for conducting scalable, high throughput selections from phage-displayed combinatorial synthetic antibody libraries against hundreds of antigens simultaneously. Using this parallel approach, we have isolated antibody fragments that exhibit high affinity and specificity for diverse antigens that are functional in standard immunoassays.

Faj-görüntülenen Sentetik Antikor Kütüphaneler itibaren Ölçeklenebilir Yüksek Verim Seçimi

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. ...

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. ...

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

Retina Mikroglial Fagositik Fonksiyonu değerlendirilmesi<em&gt; İn Vivo</em&gt; A Akış Sitometri bazlı analiz kullamlarak

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo Cell Trafficking by PET/CT

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte cell culture and 64Cu-antibody receptor targeting of the cells. In vitro evaluation of the radiolabel and non-invasive in vivo cell tracking in an animal model of an airway delayed-type hypersensitivity reaction (DTHR) by PET/CT are described.
In detail, the conjugation of a mAb with the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) is shown. Following the production of radioactive 64Cu, radiolabeling of the DOTA-conjugated mAb is described. Next, the expansion of chicken ovalbumin (cOVA)-specific CD4+ interferon (IFN)-&#947;-producing T helper cells (cOVA-TH1) and the subsequent radiolabeling of the cOVA-TH1 cells are depicted. Various in vitro techniques are presented to evaluate the effects of 64Cu-radiolabeling on the cells, such as the determination of cell viability by trypan blue exclusion, the staining for apoptosis with Annexin V for flow cytometry, and the assessment of functionality by IFN-&#947; enzyme-linked immunosorbent assay (ELISA). Furthermore, the determination of the radioactive uptake into the cells and the labeling stability are described in detail. This protocol further describes how to perform cell tracking studies in an animal model for an airway DTHR and, therefore, the induction of cOVA-induced acute airway DHTR in BALB/c mice is included. Finally, a robust PET/CT workflow including image acquisition, reconstruction, and analysis is presented.
The 64Cu-antibody receptor targeting approach with subsequent receptor internalization provides high specificity and stability, reduced cellular toxicity, and low efflux rates compared to common PET-tracers for cell labeling, e.g.64Cu-pyruvaldehyde bis(N4-methylthiosemicarbazone) (64Cu-PTSM). Finally, our approach enables non-invasive in vivo cell tracking by PET/CT with an optimal signal-to-background ratio for 48 h. This experimental approach can be transferred to different animal models and cell types with membrane-bound receptors that are internalized.

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo  Cell Trafficking by PET/CT

Following the preparation of a 64Cu-modified monoclonal antibody binding to a transgenic murine T cell receptor, T cells are radiolabeled in vivo, analyzed for viability, functionality, labeling stability and apoptosis, and adoptively transferred into mice with an airway delayed-type hypersensitivity reaction for non-invasive imaging by positron emission tomography/computed tomography (PET/CT).

tarafından Kemirgen Lenfosit Etiketleme<sup&gt; 64</sup&gt; Cu-Antikor Reseptör için hedefleme<em&gt; In Vivo</em&gt; PET / CT ile hücre Trafik

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface glycoproteins of the virus. The generation of escape variants is a powerful method in elucidating how viruses escape immune detection and in identifying critical residues required for antibody binding. Here, we describe a protocol on how to generate influenza A virus escape variants by utilizing human or murine monoclonal antibodies (mAbs) directed against the viral hemagglutinin (HA). With the use of our technique, we previously characterized critical residues required for the binding of antibodies targeting either the head or stalk of the novel avian H7N9 HA. The protocol can be easily adapted for other virus systems. Analyses of escape variants are important for modeling antigenic drift, determining single nucleotide polymorphisms (SNPs) conferring resistance and virus fitness, and in the designing of vaccines and/or therapeutics.

We describe a method by which we identify critical residues required for the binding of human or murine monoclonal antibodies that target the viral hemagglutinin of influenza A viruses. The protocol can be adapted to other virus surface glycoproteins and their corresponding neutralizing antibodies.

Grip virüsü monoklonal antikorları nötralize kaçış türevleri üretimi

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...

Measuring Influenza Neutralizing Antibody Responses to A(H3N2) Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for influenza infections. Microneutralization (MN) assays are often used to measure neutralizing antibody responses in human sera after influenza vaccination or infection. Madine Darby Canine Kidney (MDCK) cells are the commonly used cell substrate for MN assays. However, currently circulating 3C.2a and 3C.3a A(H3N2) influenza viruses have acquired altered receptor binding specificity. The MDCK-SIAT1 cell line with increased &#945;-2,6 sialic galactose moieties on the surface has proven to provide improved infectivity and more faithful replications than conventional MDCK cells for these contemporary A(H3N2) viruses. Here, we describe a MN assay using MDCK-SIAT1 cells that has been optimized to quantify neutralizing antibody titers to these contemporary A(H3N2) viruses. In this protocol, heat inactivated sera containing neutralizing antibodies are first serially diluted, then incubated with 100 TCID50/well of influenza A(H3N2) viruses to allow antibodies in the sera to bind to the viruses. MDCK-SIAT1 cells are then added to the virus-antibody mixture, and incubated for 18 - 20 h at 37 &#176;C, 5% CO2 to allow A(H3N2) viruses to infect MDCK-SIAT1 cells. After overnight incubation, plates are fixed and the amount of virus in each well is quantified by an enzyme-linked immunosorbent assay (ELISA) using anti-influenza A nucleoprotein (NP) monoclonal antibodies. Neutralizing antibody titer is defined as the reciprocal of the highest serum dilution that provides &#8805;50% inhibition of virus infectivity.

Measuring Influenza Neutralizing Antibody Responses to AH3N2 Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Influenza neutralizing antibodies correlate with protection of influenza infections. Microneutralization assays measure neutralizing antibodies in human sera and are often used for influenza human serology. We describe a microneutralization assay using MDCK-SIAT1 cells to measure neutralizing antibody titers to contemporary 3C.2a and 3C.3a A(H3N2) viruses following influenza vaccination or infection.

MDCK-SIAT1 hücreleri kullanarak antikor yanıt için Microneutralization tarafından insan Sera A(H3N2) virüsleri nötralize grip ölçme deneyi

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

Roman besleyici-Alerjik sistemi fare doğal katil hücreler Vitro seri üretim için

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor (GM-CSF)-producing T Helper (THGM) Cells

The granulocyte-macrophage-colony-stimulating factor (GM-CSF)-producing T helper (THGM) cell is a newly identified T helper cell subset that predominantly secretes GM-CSF without producing interferon (IFN)&#947; or interleukin (IL)-17 and is found to play an essential role in the autoimmune neuroinflammation. A method of isolation of naive CD4+ T cells from a single-cell suspension of splenocytes and THGM cell generation from naive CD4+ T cells would be a useful technique in the study of T cell-mediated immunity and autoimmune diseases. Here we describe a method that differentiates mouse naive CD4+ T cells into THGM cells promoted by IL-7. The outcome of the differentiation was assessed by the analysis of the cytokines expression using different techniques, including intracellular cytokine staining combined with flow cytometry, a quantitative real-time polymerase chain reaction (PCR), and enzyme-linked immunosorbent assays (ELISA). Using the THGM differentiation protocol as described here, about 55% of the cells expressed GM-CSF with a minimal expression of IFN&#945; or IL-17. The predominant expression of GM-CSF by THGM cells was further confirmed by the analysis of the expression of GM-CSF, IFN&#945;, and IL-17 at both mRNA and protein levels. Thus, this method can be used to differentiate naive CD4+ T cells to THGM cells in vitro, which will be useful in the study of THGM cell biology.

In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor GM-CSF-producing T Helper THGM Cells

Here, we present a protocol to differentiate murine granulocyte-macrophage-colony-stimulating-factor-producing T helper (THGM) cells from naive CD4+ T cells, including isolation of naive CD4+ T cells, differentiation of THGM, and analysis of differentiated THGM cells. This method can be applied to studies of the regulation and function of THGM cells.

Vitro Farklılaşma fare granülosit-makrofaj-koloni uyarıcı faktör (GM-CSF)-T yardımcı (THGM) hücreleri üreten

The granulocyte-macrophage-colony-stimulating factor (GM-CSF)-producing T helper (THGM) cell is a newly identified T helper cell subset that predominantly ...

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for macrophages. This predatory organism, similar to macrophages, employs phagocytosis to kill internalized cells. With the aid of a confocal laser-scanning microscope, images depicting interactive moments between cryptococcal cells and amoeba are captured. The resolution power of the electron microscope also helps to reveal the ultrastructural detail of cryptococcal cells when trapped inside the amoeba food vacuole. Since phagocytosis is a continuous process, quantitative data is then integrated in the analysis to explain what happens at the timepoint when an image is captured. To be specific, relative fluorescence units are read in order to quantify the efficiency of amoeba in internalizing cryptococcal cells. For this purpose, cryptococcal cells are stained with a dye that makes them fluoresce once trapped inside the acidic environment of the food vacuole. When used together, information gathered through such techniques can provide critical information to help draw conclusions on the behavior and fate of cells when internalized by amoeba and, possibly, by other phagocytic cells.

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

This paper details a protocol for preparing a co-culture of cryptococcal cells and amoebae that is studied using still, fluorescent images and high-resolution transmission electron microscope images. Illustrated here is how quantitative data can complement such qualitative information.