我々は彼らの貴重な貢献のためのK.中垣、博士石井、博士鈴木、A.山形、K. Oofusaに非常に感謝しています。

1。 sGMRαの生産と精製<ol><li>ポリメラーゼ連鎖反応（PCR）により、ヒト胎盤cDNAライブラリーからsGMRαのcDNAを増幅する。 </li><li> Hisタグ別のPCRによるPCRアンプリコンの3&#39;末端に50塩基のバキュロウイルスポリヘドリン最後の5&#39; - UTR配列と27塩基のコーディングRGSを追加。 </li><li>プラスミドpMSG1.1MGに増幅されたPCR産物を挿入します。 </li><li>カイコPND - W1株の卵にpHA3PIGヘルパーのベクトルを持つ構造psGMR/M1.1MGを注入する。 </li><li> 25蛾のリア孵化したG0幼虫℃にトランスジェニックカイコがsGMRα遺伝子を有する得るために目にMGFP発現に兄弟間やPND - W1との交配によって得られる画面のG1の胚。 </li><li> 500mmのNaClを含むリン酸緩衝液をトランスジェニックカイコの繭から組換えsGMRαを抽出し、4℃で撹拌℃で24時間のために。 </li><li>沈殿除去するために15分を20000 gでサンプルを遠心する。 </li><li>ニッケル親和性カラムに上清を適用する。 </li><li> 10mMのイミダゾール、500mmのNaClおよび20mMのリン酸ナトリウム、pH6.3、および10mmから250mmのへのイミダゾールの直線勾配で溶出するとカラムを洗浄します。プール精製sGMRαを含む画分と液（pH7.4）リン酸緩衝生理食塩水に対して透析（PBS）。 </li></ol> 2。組換えGM - CSFのビオチン化<ol><li> PBSに対して透析によってソルビトール製剤を削除します。 </li><li> ℃の氷の上で解決するために4℃で20mMの冷ナトリウムメタ過ヨウ液を1ml追加。 </li><li>暗所で氷上で30分間サンプルをインキュベートする。 </li><li> 15mmの最終濃度で溶液にグリセロールを追加。 </li><li> 100mMの酢酸ナトリウム緩衝液（pH5.5）に対してソリューションを透析</li><li> 5mmの最終濃度の溶液にビオチンヒドラジドを追加し、継続的に室温で2時間のためのソリューションを撹拌。 </li><li> PBS、pH7.4の透析によって未反応物質を取り除きます。 </li></ol> 3。無細胞GM - CSF受容体 - リガンド結合アッセイ<ol><li>一晩4モノクローナル抗ヒスチジン抗体50μlのあるコートマイクロタイタープレート℃の</li><li> PBSTの500μLで5回洗浄する。 </li><li> 4℃で3時間のためのブロッキング溶液とインキュベート℃の100μLを加える</li><li> PBSTの500μLで5回洗浄する。 </li><li>ブロッキング溶液でsGMRα50μlのを追加し、4℃で一晩インキュベート℃、 </li><li> PBSTの500μLで5回洗浄する。 </li><li>その後、GM - CSF自己抗体とビオチン標識GM - CSFの25μlのを含むサンプルの25μlのを追加。 4℃で1時間インキュベート° CsGMRα- GM - CSFの複合体を形成する。 </li><li> PBSTの500μLで5回洗浄する。 </li><li> ° C、ビオチン化GM - CSF結合sGMRαを検出するために4℃で1時間のために0.4mmのストレプトアビジンアルカリホスファターゼ50μlのを追加。 </li><li> PBSTの500μLで5回洗浄する。 </li><li>ソリューションに化学発光基質50μlのを追加し、室温で1時間インキュベートした。 </li><li>化学発光プレートリーダーを用いて、化学発光活性を検出する。 </li></ol> 4。代表的な結果： GM - CSF受容体のシグナル伝達経路の最初のステップは、細胞表面上にGM - CSF受容体αに対す​​るGM - CSFのバインディングです。 GM - CSF自己抗体が特にGM - CSFに結合し、in vitroで16,17の受容体への結合をブロックするため、我々は、自己抗体が直接的にGM - CSFに結合することによって、この最初の反応を抑制するという仮説を立てた。前述のように（文献9-15）、我々は高純度の組換えsGMRαを大量に得るために遺伝子組換えカイコ技術を適用。 唯一の単量体は還元条件下で検出されたのに対し、カイコ由来の組換えsGMRαが非還元条件下SDS - PAGEにロードされたときに、sGMRαは（図1B）、両方の単量体（45 kDa）と二量体（90 kDa）はフォームを示した、組換えsGMRαはモノマーとジスルフィド結合調光器18の混合物であることを示す。 無細胞系（図2A）を使用して、我々はGM - CSF自己抗体（図2Bおよび図2C）によるsGMRαにGM - CSF結合の阻害を評価した。これらのメソッドは、浦野らによって文献19に記述されている。増殖阻害は、密接にGM - CSF自己抗体（図3A）19の種々の濃度の結合阻害した（r = 0.988、P = 0.002）と相関していた。同様に、結合阻害は著しく成長の阻害と相関していた。結合阻害は、GM - CSF自己抗体（図3B）19によって用量依存的に増加した。別の患者からの血清IgG分画のため、これらの2つのパラメータが互いに相関した（R = 0.589、P = 0.006、図3C）。どちらの結合阻害も生育阻害は、Kd値と相関し、したがって、両方のパラメータは、結合親和性19によって影響を受けなかった。結合とgの間でデータの結果として再現性3つの異なるサンプル上の3つの独立した実験で得られたrowthの阻害を評価した。変動係数は、無細胞系は、バイオアッセイ（表1）のそれより優れていることを示した。 <img src="/files/ftp_upload/2742/2742fig1.jpg" alt="図1" /> 図1トランスジェニックカイコを用いたsGMRαの生産のための手順のフローチャート。形質転換ベクター9-11 A）構造。還元および非還元条件下での精製sGMRαのB）SDS - PAGEとクマシーブリリアントブルー染色。 sGMRα、可溶性のGM - CSF受容体α、MGFP、モンスター緑色蛍光タンパク質、BmNPVpol5&#39; - UTR、 カイコ核多角体病ウイルス多角体の5&#39; -非翻訳領域配列; SV40ポリA、SV40ポリAシグナル配列、P 3xP3、3xP3プロモーター、P SER1、SER1プ ​​ロモーター; fibLポリA、フィブロインL鎖ポリAシグナル配列、HR3、 カイコ核多角体病ウイルスHR3エンハンサー。 <img src="/files/ftp_upload/2742/2742fig2.jpg" alt="図2" /> 図2無細胞アッセイ系のスキーム。 A）カイコで生産さsGMRαを用いた競合結合アッセイは。 B）中和と非中和抗体の効果は、無細胞系による結合阻害について。 C）様々な中和抗体の濃度と非中和抗体との間の結合阻害の差は。 GM - CSF、顆粒球マクロファージコロニー刺激因子、sGMRα、水溶性のGM - CSF受容体α、彼の、RGS - Hisタグ、AP、アルカリホスファターゼ。 <img src="/files/ftp_upload/2742/2742fig3.jpg" alt="図3" /> 図3。GM - CSFモノクローナル抗体または自己免疫PAP患者からの血清IgG画分の影響によりsGMRαにGM - CSF結合阻害。 GM - CSF自己抗体による結合阻害や増殖阻害との間のA）の関係。 B）GM - CSF自己抗体の種々の濃度の結合阻害。血清IgG画分19重量％の結合阻害と％の成長阻害のIC 50との間のC）の関係。 IC 50、50％阻害濃度、GM - CSF、顆粒球マクロファージコロニー刺激因子。 <table border="1"><tbody><tr><td>サンプル</td><td> A </td><td> B </td><td> C </td></tr><tr><td colspan="4">アッセイ間</td></tr><tr><td> ＃決定の</td><td> 3 </td><td> 3 </td><td> 3 </td></tr><tr><td>平均値（％結合阻害） </td><td> 61.6 </td><td> 63.8 </td><td> 69.7 </td></tr><tr><td>平均値（％増殖阻害） </td><td> 21.0 </td><td> 73.4 </td><td> 82.8 </td></tr><tr><td>変動係数（％）（％結合阻害） </td><td> 6.0 </td><td> 5.6 </td><td> 8.3 </td></tr><tr><td>変動係数（％）（％増殖阻害） </td><td> 64.4 </td><td> 18.3 </td><td> 10.4 </td></tr></tbody></table>両方のアッセイは、5 ng / mlのGM - CSFのとGM - CSF自己抗体と同等の濃度で行った。 表1 3つの独立した実験で得られたパーセントの結合と成長の阻害との間の変動係数の比較。

<ol>
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無細胞アッセイは、優れた再現性と速さとGM - CSF自己抗体の中和能を推定する。 GM - CSF自己抗体または患者の血清IgG分画による結合阻害は、このアッセイにより評価した。データは、無細胞アッセイの結合阻害とそれぞれTF - 1細胞​​を用いてバイオアッセイの増殖阻害との間の相関を示した。バイオアッセイは、広く利用され、しかし別の施設と我々はこの新しいシステムを使用すること?...

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

背景：これまで、我々はその中和能を示したが、GM - CSF自己抗体の濃度は、自己免疫性肺肺胞タンパク症（PAP）1-3患者の疾患の重症度と相関していたではない。肺におけるGM - CSFの生物活性の廃止は、4,5自己免疫PAPの可能性が高い原因であるとして、それはPAPと各患者の病気の重症度を評価するためのGM - CSF自己抗体の中和能を測定するために期待される。 今までは、GM - CSF自己抗体の中和能は、ヒト骨髄細胞またはGM - CSF 6月8日で刺激TF - 1細胞の増殖阻害を評価することによって評価されている。バイオアッセイ系では、しかしながら、一定の条件で細胞を維持するための技術的な問題に起因する別の研究室からのデータを、比較するだけでなく、信頼性の高いデータを得るためにしばしば問題になります。 目的：無細胞受容体結合アッセイを用いて細胞表面にGM - CSF受容体に結合するGM - CSFを模倣する。 方法：トランスジェニックカイコ技術は、高純度9月13日に組換え可溶性GM - CSF受容体α（sGMRα）のための大規模な量を得るために適用した。組換えsGMRαは、絹のタンパク質に融合されることなく、絹の糸の親水性のセリシン層に含まれていた、従って、我々は容易に中性の水溶液14,15との良好な純度で繭から抽出することができます。幸いなことに、GM - CSFとの結合に不可欠なオリゴ糖の構造は、他の昆虫や酵母によって生成されるものよりも人間のsGMRαの構造に、より類似しています。 結果：無細胞アッセイ系sGMRαを使用しては高い可塑性と信頼性でデータが得られた。 sGMRαにGM - CSF結合を用量依存sGMRαを使って、新しい無細胞アッセイ系は、中和活性の測定のためのより有用であることを示す、TF - 1細胞​​を用いたバイオアッセイと同様の方法でポリクローナルGM - CSF自己抗体によって阻害されたTF - 1細胞​​またはヒト骨髄細胞を用いたバイオアッセイ系よりGM - CSF自己抗体の。 結論：我々はGM - CSF自己抗体の中和能を定量化する無細胞アッセイを確立した。

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

我々は、受容体への顆粒球マクロファージコロニー刺激因子（GM - CSF）の結合を推定するために、無細胞受容体結合アッセイをデザインしました。それは、優れた再現性とGM - CSF自己抗体による可溶性GM - CSF受容体αに結合するビオチン化GM - CSFの競合的阻害を評価することを可能にします。

組換え可溶性GM - CSF受容体を用いてGM - CSF抗体の中和能を推定する無細胞アッセイシステム

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

Research

JoVE Journal

Immunology and Infection

12.6K ビュー.  Niigata University Medical and Dental Hospital. 我々は、受容体への顆粒球マクロファージコロニー刺激因子（GM - CSF）の結合を推定するために、無細胞受容体結合アッセイをデザインしました。それは、優れた再現性とGM - CSF自己抗体による可溶性GM - CSF受容体αに結合するビオチン化GM - CSFの競合的阻害を評価することを可能にします。

ビデオ: 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.

ヒト白血球と血小板の単離と評価のためのプロトコル：生体エネルギーと酸化バースト

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

免疫・感染学

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.

ファージディスプレイされた合成抗体ライブラリーから、スケーラブルな高スループットの選択

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.

マウスの骨髄からGM-CSF由来肺胞のようなマクロファージや樹状細胞の生成および同定

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.

網膜ミクログリアの貪食機能を評価します<em&gt;インビボ</em&gt;フローサイトメトリーベースのアッセイを使用しました

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).

マウスリンパ球標識による<sup&gt; 64</sup以下のためのターゲット設定&gt;のCu-抗体の受容体<em&gt;インビボ</em&gt; PET / CTによる細胞輸送

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.

エスケープ中和抗体でインフルエンザ ウイルスの亜種の世代

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.

試金を使用して SIAT1 MDCK 細胞のインフルエンザ a (h3n2) ウイルス中和によるひと血清中の抗体を中和することを測定

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.

生体外でマウスのナチュラル キラー細胞の大量生産のため新規無料の送り装置システム

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.

体外分化マウス顆粒球マクロファージ ・ コロニー刺激因子 (GM-CSF)-(THGM) ヘルパー T 細胞の生産

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.