نحن ممتنون جدا لNakagaki K. ، الدكتور إيشي H. ، الدكتور سوزوكي K. ، ياماغاتا A. ، K. Oofusa لما قدموه من مساهمات قيمة.

1. إنتاج وتنقية sGMRα <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> [كدنا] تضخيم من sGMRα من المشيمة البشرية [كدنا] مكتبة عن طريق تفاعل البلمرة المتسلسل (PCR). </li><li style=";text-align:right;direction:rtl"> إضافة قاعدة 5&#39; - 50 UTR تسلسل نهاية polyhedrin الفيروسة العصوية وRGS قاعدة 27 - الترميز صاحب العلامة لانهاء 3 &#39;إلى آخر amplicon PCR PCR. </li><li style=";text-align:right;direction:rtl"> إدراج منتج PCR تضخيمها الى pMSG1.1MG البلازميد. </li><li style=";text-align:right;direction:rtl"> حقن psGMR/M1.1MG بناء ناقلات مع مساعد pHA3PIG في بويضات من سلالة PND - W1 دودة القز. </li><li style=";text-align:right;direction:rtl"> مؤخرة تحاك ليرقات العث G0 عند 25 درجة مئوية. أجنة G1 الشاشة التي حصلت عليها التزاوج بين الأشقاء أو مع PND - W1 للتعبير MGFP في أعين المعدلة وراثيا للحصول على دودة القز التي تحمل الجينات sGMRα. </li><li style=";text-align:right;direction:rtl"> استخراج المؤتلف sGMRα من شرانق دود القز وراثيا للفوسفات مع العازلة التي تحتوي على كلوريد الصوديوم ويقلب 500mM عند 4 درجات مئوية عن 24H. </li><li style=";text-align:right;direction:rtl"> أجهزة الطرد المركزي في عينة ز 20000 ل15min لإزالة رواسب. </li><li style=";text-align:right;direction:rtl"> تطبيق طاف إلى عمود النيكل تقارب. </li><li style=";text-align:right;direction:rtl"> غسل العمود مع إيميدازول 10MM ، كلوريد الصوديوم 500mM و 20mm فوسفات الصوديوم ، pH6.3 ، وأزل مع التدرج الخطي للإيميدازول من 10mm إلى 250mM. تجمع الكسور التي تحتوي على sGMRα المنقى وdialyze ضد الفوسفات مخزنة المالحة (pH7.4) (PBS). </li></ol> 2. Biotinylation من المؤتلف GM - CSF <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إزالة إعداد السوربيتول ضد غسيل الكلى عن طريق برنامج تلفزيوني. </li><li style=";text-align:right;direction:rtl"> إضافة 1ml من 20mM الباردة حل التلوي بريودات الصوديوم عند 4 درجة مئوية إلى الحل على الجليد. </li><li style=";text-align:right;direction:rtl"> نموذج لاحتضان 30min على الجليد في الظلام. </li><li style=";text-align:right;direction:rtl"> إضافة الجلسرين إلى الحل في التركيز النهائي لل15mM. </li><li style=";text-align:right;direction:rtl"> Dialyze الحل ضد 100mM خلات الصوديوم العازلة (pH5.5) </li><li style=";text-align:right;direction:rtl"> إضافة البيوتين هيدرازيد إلى الحل في التركيز النهائي لل5mM وتحرض باستمرار على حل ل2H في درجة حرارة الغرفة. </li><li style=";text-align:right;direction:rtl"> إزالة المواد غير رد فعل من قبل غسيل الكلى ضد برنامج تلفزيوني ، pH7.4. </li></ol> 3. خلية خالية GM - CSF مستقبلات يجند ملزم مقايسة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> معطف لوحة microtiter مع 50μl من الأجسام المضادة وحيدة النسيلة المضادة للpolyHistidine بين عشية وضحاها في 4 درجات مئوية. </li><li style=";text-align:right;direction:rtl"> تغسل خمس مرات مع 500μl من PBST. </li><li style=";text-align:right;direction:rtl"> إضافة 100μl حل الحجب واحتضان ل3H في 4 درجات مئوية. </li><li style=";text-align:right;direction:rtl"> تغسل خمس مرات مع 500μl من PBST. </li><li style=";text-align:right;direction:rtl"> إضافة 50μl من sGMRα في حل الحجب واحتضان بين عشية وضحاها في 4 درجات مئوية. </li><li style=";text-align:right;direction:rtl"> تغسل خمس مرات مع 500μl من PBST. </li><li style=";text-align:right;direction:rtl"> إضافة 25μl من العينات تحتوي على GM - CSF الأجسام المضادة ثم 25μl من biotinylated GM - CSF. لاحتضان 1H في 4 درجات مئوية لتشكيل sGMRα - GM - CSF المعقدة. </li><li style=";text-align:right;direction:rtl"> تغسل خمس مرات مع 500μl من PBST. </li><li style=";text-align:right;direction:rtl"> إضافة 50μl من الفوسفاتيز القلوية streptavidin 0.4mM ل1hour في 4 درجات مئوية للكشف عن biotinylated GM - CSF sGMRα ملزمة. </li><li style=";text-align:right;direction:rtl"> تغسل خمس مرات مع 500μl من PBST. </li><li style=";text-align:right;direction:rtl"> إضافة 50μl من ركيزة chemiluminescent إلى الحل والمحتضنة ل1H في درجة حرارة الغرفة. </li><li style=";text-align:right;direction:rtl"> لمعان كيميائي باستخدام قارئ لوحة ، وكشف عن نشاط لمعان كيميائي. </li></ol> 4. ممثل النتائج : الخطوة الأولى في مسار مستقبلات GM - CSF نقل الإشارة هو ملزم GM - CSF لمستقبلات ألفا GM - CSF على سطح الخلية. لأن الأجسام المضادة GM - CSF تربط تحديدا GM - CSF وكتلة ملزم لمستقبلات 16،17 في المختبر ، ونحن افترضنا أن هذه الأجسام المضادة تمنع أول رد فعل من قبل ملزمة مباشرة إلى GM - CSF. كما هو موضح سابقا (المرجع 9-15) ، طبقنا المعدلة وراثيا دودة القز التكنولوجيا للحصول على كمية كبيرة من sGMRα المؤتلف مع درجة نقاوة عالية. وأظهرت sGMRα عندما تم تحميل دودة القز المستمدة sGMRα المؤتلف على SDS - PAGE تحت ظروف غير الحد ، سواء موحودي (45 كيلو دالتون) ومثنوي (90 كيلو دالتون) الأشكال ، في حين تم الكشف عن النموذج الوحيد في ظل ظروف موحودي الحد (1B الشكل) ، مشيرا الى ان sGMRα المؤتلف وكان مزيجا من مونومرات وثاني كبريتيد مرتبطة المخفتات 18. باستخدام نظام خالية من الخلايا (الشكل 2A) ، ونحن قيمت تثبيط GM - CSF ملزمة لsGMRα من قبل جنرال موتورز - CSF الأجسام المضادة (الشكل 2B 2C والشكل). وقد وصفت هذه الأساليب في اشارة بواسطة أورانو 19 آخرون. وترتبط بشكل وثيق مع تثبيط نمو تثبيط ملزم (ص = 0.988 ، P = 0.002) بتركيزات مختلفة من الأجسام المضادة GM - CSF (الشكل 3A) 19. وبالمثل ، فقد ارتبط بشكل ملحوظ تثبيط ملزم مع تثبيط النمو. وتثبيط ملزم زادت بطريقة تعتمد على الجرعة التي كتبها GM - CSF الأجسام المضادة (الشكل 3B) 19. هذه كانت مرتبطة المعلمتين عن كسور مفتش المصل من المرضى المختلفة بعضها البعض (ص = 0.589 ع = 0.006 ، الشكل 3C). لا تثبيط ملزم ولا تثبيط النمو يرتبط بقيم دينار كويتي ، وبالتالي كل من المعلمات لم تتأثر ملزمة تقارب 19. استنساخ بالتالي ملزمة للبيانات بين وزوقد تم تقييم الحصول عليها عن طريق تثبيط rowth ثلاث تجارب مستقلة على ثلاث عينات مختلفة. وأشار إلى أن معامل الاختلاف في منظومة خالية من الخلايا كان أكثر من ممتازة من ذلك الأحيائي (الجدول 1). <img src="/files/ftp_upload/2742/2742fig1.jpg" alt="الشكل 1" /> الشكل 1. مخطط تدفق الإجراء لإنتاج المعدلة وراثيا باستخدام sGMRα دودة القز. أ) هياكل ناقلات التحول 9-11. B) SDS - PAGE وCoomassie بريليانت الأزرق تلطيخ لتنقية sGMRα تحت تخفيض والحد من ظروف عدم. sGMRα ، قابل للذوبان GM - CSF مستقبلات ألفا ؛ MGFP ، بروتين فلوري الوحش الأخضر ؛ BmNPVpol5&#39; - UTR ، 5&#39; - تسلسل غير مترجمة المنطقة من الأسلحة النووية Bombyx موري polyhedrin فيروس polyhedrosis ؛ SV40 polyA ، SV40 تسلسل إشارة polyA ؛ ف 3xP3 ، 3xP3 المروج ؛ P ser1 ، ser1 المروج ؛ fibL polyA ، فبروين إشارة polyA L - سلسلة تسلسل ؛ hr3 ، Bombyx موري فيروس polyhedrosis النووية hr3 محسن. <img src="/files/ftp_upload/2742/2742fig2.jpg" alt="الشكل 2" /> الشكل 2. مخطط للنظام مقايسة خالية من الخلايا. أ) مقايسة تنافسية ملزمة باستخدام sGMRα التي تنتجها دودة القز. ب) تأثير تحييد الأجسام المضادة لإبطال مفعولها وغير على تثبيط ملزمة خالية من الخلايا النظام. C) والفرق بين من تثبيط ملزم تركيزات مختلفة من الأجسام المضادة والأجسام المضادة غير لإبطال مفعولها. GM - CSF ، محببة ، بلعم مستعمرة عامل تحفيز ؛ ، قابل للذوبان sGMRα GM - CSF مستقبلات ألفا ؛ بلده ، RGS - - صاحب العلامة ؛ أ ب ، و الفوسفاتيز القلوية. <img src="/files/ftp_upload/2742/2742fig3.jpg" alt="الشكل 3" /> الشكل 3. GM - CSF ملزمة لتثبيط تأثير sGMRα بواسطة GM - CSF النسائل المتعددة أو الأجسام المضادة في مصل الدم كسور مفتش من المرضى الذين يعانون من PAP الذاتية. أ) العلاقة بين تثبيط ملزمة وتثبيط النمو GM - CSF الأجسام المضادة. B) تثبيط ملزم بتركيزات مختلفة من الأجسام المضادة GM - CSF. ج) العلاقة بين IC 50 في المئة لتثبيط ملزمة وتثبيط النمو في المئة من الكسور مفتش المصل 19. IC 50 ، 50 ٪ تركيز المثبطة ؛ GM - CSF ، محببة ، بلعم مستعمرة عامل تحفيز. <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> عينة </td><td> A </td><td> B </td><td> C </td></tr><tr><td colspan="4"> بين مقايسة </td></tr><tr><td> # من القرارات </td><td> 3 </td><td> 3 </td><td> 3 </td></tr><tr><td> يعني القيمة (٪ تثبيط ملزم) </td><td> 61.6 </td><td> 63.8 </td><td> 69.7 </td></tr><tr><td> يعني القيمة (٪ تثبيط النمو) </td><td> 21.0 </td><td> 73.4 </td><td> 82.8 </td></tr><tr><td> معامل الاختلاف (٪) (٪ تثبيط ملزم) </td><td> 6.0 </td><td> 5.6 </td><td> 8.3 </td></tr><tr><td> معامل الاختلاف (٪) (٪ تثبيط النمو) </td><td> 64.4 </td><td> 18.3 </td><td> 10.4 </td></tr></tbody></table> وأجريت فحوصات على حد سواء في 5 نانوغرام / مل من جنرال موتورز - CSF وتركيز يساوي GM - CSF الأجسام المضادة. الجدول 1. مقارنة الاختلافات بين معامل ملزم في المئة ونمو الموانع الحصول عليها من خلال ثلاث تجارب مستقلة.

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قدرت مقايسة خالية من الخلايا تحييد قدرة جنرال موتورز - CSF الأجسام المضادة مع استنساخ ممتازة وسرعة. تم تقييم تثبيط ملزم من قبل جنرال موتورز - CSF الأجسام المضادة أو المريض المصل الكسور مفتش بواسطة هذا الاختبار. وأظهرت البيانات وجود ارتباط بين تثبيط ملزم للمقايسة خالية م?...

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

الخلفيات : في السابق ، ونحن أثبتت أن القدرة على تحييد لكنه لا يرتبط تركيز الأجسام المضادة GM - CSF مع شدة المرض في المرضى الذين يعانون من المناعة الذاتية البروتيني السنخي الرئوي (PAP) 1-3. وإلغاء النشاط الحيوي GM - CSF في الرئة هو السبب المرجح لPAP 4،5 المناعة الذاتية ، فهو يبشر بالخير لقياس قدرة جنرال موتورز تحييد الأجسام المضادة - CSF لتقييم شدة المرض في كل مريض مع PAP. حتى الآن ، وقد تم تقييم قدرة جنرال موتورز تحييد الأجسام المضادة - CSF من خلال تقييم تثبيط نمو خلايا نخاع العظام أو خلايا TF - 1 حفز مع GM - CSF 6-8. في النظام الأحيائي ، ومع ذلك ، غالبا ما يكون مشكلة للحصول على بيانات موثوق بها وكذلك للمقارنة بين البيانات المأخوذة من مختلف المختبرات ، وذلك بسبب الصعوبات التقنية في الحفاظ على الخلايا في حالة ثابتة. الهدف : محاكاة GM - CSF ملزم لمستقبلات GM - CSF على سطح الخلية باستخدام خالية من الخلايا مستقبلات ملزمة مقايسة. تم تطبيق تكنولوجيا المعدلة وراثيا دودة القز من أجل الحصول على مبلغ كبير بالنسبة لالمؤتلف مستقبلات ألفا للذوبان GM - CSF (sGMRα) مع درجة نقاوة عالية 9-13 : طرق. وكان يرد sGMRα المؤتلف في الطبقات المائية سيريسين من الخيوط الحريرية دون تنصهر للبروتينات الحرير ، وبالتالي ، يمكننا بسهولة استخراج من الشرانق في الطهارة جيدة مع المحاليل المائية المحايدة 14،15. لحسن الحظ ، فإن الهياكل oligosaccharide ، التي هي حيوية للربط مع GM - CSF ، أكثر مشابهة للهياكل sGMRα الإنسان من تلك التي تنتجها الحشرات الأخرى أو الخمائر. النتائج : نظام مقايسة خالية من الخلايا باستخدام sGMRα أسفرت البيانات مع مرونة وموثوقية عالية. وكان GM - CSF ملزمة لsGMRα الجرعة بشكل تابع تثبطها الأجسام المضادة GM - CSF النسائل المتعددة بطريقة مشابهة لما الأحيائي باستخدام الخلايا TF - 1 ، مشيرا إلى أن لدينا نظام جديد خالية من الخلايا باستخدام مقايسة sGMRα هو أكثر فائدة لقياس النشاط تحييد GM - CSF من الأجسام المضادة من النظام الأحيائي باستخدام TF - 1 خلية أو خلايا نخاع العظام. الاستنتاجات : نحن إنشاء مقايسة خالية من الخلايا قياس قدرة جنرال موتورز تحييد الأجسام المضادة - CSF.

Watch this Scientific Journal Video about A Cell Free Assay System Estimating the Neutralizing Capacity of GM-CSF Antibody using Recombinant Soluble GM-CSF Receptor at JoVE.com

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

قمنا بتصميم تجربة خالية من الخلايا مستقبلات ملزمة من أجل تقدير الربط عامل تحفيز مستعمرة محببة - البلاعم (GM - CSF) إلى المستقبلات. انها تمكننا من تقييم التثبيط التنافسي للbiotinylated GM - CSF ملزم لمستقبلات ألفا للذوبان GM - CSF - CSF من قبل جنرال موتورز مع الأجسام المضادة استنساخ ممتازة.

نظام خلية الفحص مجاني تقدير تحييد القدرات من GM - CSF جسم باستخدام المؤتلف ذوبان GM - CSF مستقبلات

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

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JoVE Journal

Immunology and Infection

12.6K Views.  Niigata University Medical and Dental Hospital. قمنا بتصميم تجربة خالية من الخلايا مستقبلات ملزمة من أجل تقدير الربط عامل تحفيز مستعمرة محببة - البلاعم (GM - CSF) إلى المستقبلات. انها تمكننا من تقييم التثبيط التنافسي للbiotinylated GM - CSF ملزم لمستقبلات ألفا للذوبان GM - CSF - CSF من قبل جنرال موتورز مع الأجسام المضادة استنسا?...

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.

الطاقة الحيوية والأكسدة انفجار: بروتوكولات لعزل وتقييم الكريات البيضاء والصفائح الدموية البشرية

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

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.

تحجيم إنتاجية عالية التحديد من المكتبات الأجسام المضادة الاصطناعية-عرض فاجي

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.

جيل وتحديد GM-CSF المشتقة مثل السنخية الضامة والخلايا الجذعية من الفأر نخاع العظم

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.

تقييم الشبكية دبقية أكلة وظيفة<em&gt; في فيفو</em&gt; باستخدام الفحص القائمة على الخلوي التدفق

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).

الفئران اللمفاويات وسمها من قبل<sup&gt; 64</sup&gt; النحاس والأجسام المضادة مستقبلات استهداف ل<em&gt; في فيفو</em&gt; الاتجار خلية بواسطة PET / ط م

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.

جيل متغيرات الهروب من تحييد الأجسام المضادة فيروس الإنفلونزا

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.

قياس الإنفلونزا تحييد ردود الأجسام المضادة للفيروسات A(H3N2) في الأمصال البشرية قبل درياقي فحوصات باستخدام خلايا مدكك-SIAT1

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.

نظام تغذية خالية رواية للإنتاج الضخم للخلايا القاتلة الطبيعية مورين في المختبر

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.

في المختبر التفريق بين الماوس المحببات-بلعم-مستعمرة-حفز عامل (GM-CSF)-إنتاج خلايا مساعد (GMحتي) T

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.