אנו מודים מאוד ק Nakagaki, ד&quot;ר ח אישיאי, ד&quot;ר ק &#39;סוזוקי, א Yamagata, ק Oofusa תרומות יקר שלהם.

1. הפקת וטיהור של sGMRα <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> Amplify cDNA של sGMRα מספריית cDNA השליה האנושית על ידי שרשרת התגובה פולימראז (PCR). </li><li style=";text-align:right;direction:rtl"> הוסף 50 בסיס 5&#39;-UTR רצף של סוף polyhedrin baculovirus ו 27 RGS בסיס קידוד תג-שלו עד הסוף &quot;3 עד amplicon PCR PCR על ידי אחר. </li><li style=";text-align:right;direction:rtl"> הכנס את מוצר מוגבר PCR pMSG1.1MG לתוך פלסמיד. </li><li style=";text-align:right;direction:rtl"> להזריק את psGMR/M1.1MG לבנות עם וקטור עוזר pHA3PIG לתוך הביצים של זן PND-W1 תולעי משי. </li><li style=";text-align:right;direction:rtl"> אחורי בקעו G0 זחלי עש אל על 25 ° C. G1 מסך עוברים שהושגו ההזדווגות בין אחים או עם PND-W1 לביטוי MGFP בעיניים להשיג מהונדס תולעי משי נושאות את הגן sGMRα. </li><li style=";text-align:right;direction:rtl"> תמצית רקומביננטי sGMRα מתוך פקעות של תולעי משי מהונדס עם חיץ, פוספט המכיל 500mm NaCl ומערבבים על 4 מעלות צלזיוס למשך 24 שעות. </li><li style=";text-align:right;direction:rtl"> צנטריפוגה מדגם ב 20000 g עבור 15min להסיר משקעים. </li><li style=";text-align:right;direction:rtl"> החל supernatant לעמודה ניקל זיקה. </li><li style=";text-align:right;direction:rtl"> לשטוף את העמודה עם imidazole 10mm, 500mm NaCl פוספט נתרן 20mm, pH6.3, ו elute עם שיפוע ליניארי של imidazole מ 10mm ל 250mm. בריכת שברים המכיל את sGMRα מטוהרים dialyze נגד פוספט שנאגרו מלוחים (pH7.4) (PBS). </li></ol> 2. Biotinylation רקומביננטי של GM-CSF <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הסר הכנה סורביטול ידי דיאליזה נגד PBS. </li><li style=";text-align:right;direction:rtl"> הוסף 1ml של 20mm פתרון קר נתרן מטא periodate ב 4 ° C לפתרון על הקרח. </li><li style=";text-align:right;direction:rtl"> דגירה המדגם עבור 30 דקות על הקרח בחושך. </li><li style=";text-align:right;direction:rtl"> הוסף גליצרול לפתרון בריכוז הסופי של 15mm. </li><li style=";text-align:right;direction:rtl"> Dialyze הפתרון נגד המאגר אצטט 100mm נתרן (pH5.5) </li><li style=";text-align:right;direction:rtl"> הוסף hydrazide ביוטין לפתרון בריכוז הסופי של 5mm ברציפות להתסיס את הפתרון עבור 2h בטמפרטורת החדר. </li><li style=";text-align:right;direction:rtl"> הסר את החומר הלא הגיבו דיאליזה נגד PBS, pH7.4. </li></ol> 3. תא ללא GM-CSF קולטן ליגנד מחייב assay <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מעיל microtiter צלחת עם 50μl של נוגדנים חד שבטיים אנטי polyHistidine לילה בשעה 4 ° C. </li><li style=";text-align:right;direction:rtl"> לשטוף חמש פעמים עם 500μl של PBST. </li><li style=";text-align:right;direction:rtl"> הוסף 100μl פתרון חסימת דגירה עבור 3h ב 4 ° C. </li><li style=";text-align:right;direction:rtl"> לשטוף חמש פעמים עם 500μl של PBST. </li><li style=";text-align:right;direction:rtl"> הוסף 50μl של sGMRα בפתרון חסימת דגירה לילה בשעה 4 ° C. </li><li style=";text-align:right;direction:rtl"> לשטוף חמש פעמים עם 500μl של PBST. </li><li style=";text-align:right;direction:rtl"> הוסף 25μl של דגימות המכילות GM-CSF autoantibody ואז 25μl biotinylated של GM-CSF. דגירה עבור 1h ב 4 ° C כדי ליצור sGMRα-GM-CSF מורכבים. </li><li style=";text-align:right;direction:rtl"> לשטוף חמש פעמים עם 500μl של PBST. </li><li style=";text-align:right;direction:rtl"> הוסף 50μl של phosphatase streptavidin 0.4mM אלקליין עבור 1hour ב 4 ° C כדי לזהות את biotinylated GM-CSF מחויב sGMRα. </li><li style=";text-align:right;direction:rtl"> לשטוף חמש פעמים עם 500μl של PBST. </li><li style=";text-align:right;direction:rtl"> הוסף 50μl של המצע chemiluminescent לפתרון וטופחו במשך 1h בטמפרטורת החדר. </li><li style=";text-align:right;direction:rtl"> באמצעות קורא צלחת chemiluminescence, לזהות את פעילות chemiluminescence. </li></ol> 4. נציג תוצאות: הצעד הראשון במסלול GM-CSF אות קולטן התמרה הוא מחייב של GM-CSF אלפא GM-CSF קולטן על פני התא. בגלל GM-CSF autoantibody במיוחד נקשר GM-CSF ולחסום מחייב שלו לקולטן במבחנה 16,17, אנו משערים כי נוגדנים עצמיים לעכב את התגובה הראשונה על ידי ישירות מחייב GM-CSF. כפי שתואר קודם לכן (Ref. 15/09), אנו ליישם טכנולוגיה מהונדס תולעי משי כדי להשיג כמות גדולה של sGMRα רקומביננטי עם טוהר גבוהה. כאשר תולעי משי הנגזרות sGMRα רקומביננטי הועמס על PAGE-SDS תחת אי - הפחתת התנאים, הראה sGMRα הן monomeric (45 KDA) ו dimeric (90 KDA) טפסים, בעוד רק את הטופס monomeric זוהה בתנאים צמצום (איור 1B) , המציין sGMRα רקומביננטי היתה תערובת של מונומרים ו דיסולפיד צמודות dimmers 18. שימוש במערכת תא חופשי (איור 2 א), אנו העריכו את עיכוב של GM-CSF מחייב sGMRα על ידי GM-CSF נוגדנים עצמיים (איור 2B ו איור. 2C). שיטות אלה תוארו על ידי התייחסות 19 Urano et al. עיכוב הצמיחה היתה בקורלציה הדוק עם עיכוב מחייב (r = 0.988, p = 0.002) בריכוזים שונים של autoantibody GM-CSF (איור 3A) 19. כמו כן, עיכוב הכריכה היה בקורלציה משמעותית עם עיכוב הצמיחה. עיכוב מחייב גדל באופן תלוי מינון על ידי GM-CSF נוגדנים עצמיים (איור 3 ב) 19. אלו שני פרמטרים עבור שברים סרום IgG מחולים שונים היו מתואמים זה לזה (r = 0.589 p = 0.006, איור. 3C). לא עיכוב ולא מחייב עיכוב גדילה בקורלציה עם ערכים Kd, ולכן הפרמטרים הן לא הושפעו על ידי זיקה מחייבת 19. כתוצאה מכך שחזור של נתונים בין מחייב gעיכוב rowth שהושג באמצעות שלושה ניסויים עצמאיים על שלושה מדגמים שונים הוערך. מקדם השונות ציינו כי מערכת תא חופשי היה מצוין יותר מזה של המבדק (לוח 1). <img src="/files/ftp_upload/2742/2742fig1.jpg" alt="איור 1" /> באיור 1. תרשים זרימה של תהליך הייצור של sGMRα מהונדס באמצעות תולעי משי. א) מבנים של וקטורים שינוי 9-11. ב) SDS-PAGE ו Coomassie מבריק מכתים כחול של sGMRα מטוהרים תחת צמצום ולא הפחתת התנאים. sGMRα, מסיסים GM-CSF קולטני אלפא; MGFP, מפלצת חלבון פלואורסצנטי ירוק; BmNPVpol5&#39;-UTR, 5&#39;-מתורגמים רצף של האזור Bombyx מורי גרעיני polyhedrosis וירוס polyhedrin; SV40 פולה, פולה SV40 רצף האות; P 3xP3, 3xP3 האמרגן, P ser1, ser1 מקדם; fibL פולה, fibroin L-שרשרת רצף פולה האות; hr3, Bombyx מורי גרעיני polyhedrosis וירוס hr3 משפר. <img src="/files/ftp_upload/2742/2742fig2.jpg" alt="איור 2" /> איור 2. Scheme של מערכת תא ללא assay. א) assay מחייב תחרותי באמצעות sGMRα המיוצר על ידי טוואי המשי. ב) השפעת נוגדנים מנטרלים ולא נטרול על עיכוב מחייב ידי מערכת התא חינם. ג) ההבדל של עיכוב מחייב בין ריכוזים שונים של נוגדנים מנטרלים ולא נוגדנים מנטרלים. GM-CSF, granulocyte-macrophage המושבה גורם מגרה; sGMRα, מסיסים GM-CSF קולטני אלפא;, שלו RGS שלו, תג: AP, phosphatase אלקליין. <img src="/files/ftp_upload/2742/2742fig3.jpg" alt="איור 3" /> איור 3. GM-CSF עיכוב מחייב sGMRα על ידי השפעה של GM-CSF נוגדנים polyclonal או שברים סרום IgG מחולים עם PAP אוטואימוניות. א) הקשר בין עיכוב מחייב עיכוב גדילה על ידי GM-CSF autoantibody. ב) עיכוב עקידת בריכוזים שונים של GM-CSF autoantibody. ג) הקשר בין IC 50 עבור עיכוב אחוז מחייב צמיחה עיכוב אחוז על ידי שברים IgG בסרום 19. IC 50, ריכוז מעכבות 50%, GM-CSF, granulocyte-macrophage המושבה גורם מגרה. <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> מדגם </td><td> </td><td> ב </td><td> ג </td></tr><tr><td colspan="4"> Inter-assay </td></tr><tr><td> # של קביעות </td><td> 3 </td><td> 3 </td><td> 3 </td></tr><tr><td> ערך ממוצע (% עיכוב מחייב) </td><td> 61.6 </td><td> 63.8 </td><td> 69.7 </td></tr><tr><td> ערך ממוצע (צמיחה עיכוב%) </td><td> 21.0 </td><td> 73.4 </td><td> 82.8 </td></tr><tr><td> מקדם השונות (%) (% עיכוב מחייב) </td><td> 6.0 </td><td> 5.6 </td><td> 8.3 </td></tr><tr><td> מקדם השונות (%) (צמיחה עיכוב%) </td><td> 64.4 </td><td> 18.3 </td><td> 10.4 </td></tr></tbody></table> מבחני שניהם בוצעו 5 ng / ml של GM-CSF ו ריכוז שווה GM-CSF autoantibody. טבלה 1. השוואה בין מקדם של וריאציות בין מחייב אחוזים עכבות הצמיחה שהושג באמצעות שלושה ניסויים עצמאיים.

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Assay תא ללא העריכו את יכולת נטרול של GM-CSF נוגדנים עצמיים עם שחזור המהירות מעולה. עיכוב הכריכה על ידי GM-CSF או שברים נוגדנים עצמיים בסרום של החולה IgG הוערך על ידי assay זה. הנתונים הראו מתאם בין עיכוב המחייב של assay התא חינם ועל עיכוב הצמיחה של המבדק באמצעות TF-1 תאים, בהתאמה. המבד?...

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

רקע: בעבר, הראינו כי יכולת נטרול אך לא את הריכוז של GM-CSF autoantibody היתה בקורלציה עם חומרת המחלה בחולים עם proteinosis אוטואימוניות מכתשיים ריאתי (PAP) 1-3. כפי וביטול bioactivity GM-CSF בריאה היא הגורם סביר PAP אוטואימוניות 4,5, הוא מבטיח למדוד את יכולת נטרול של GM-CSF נוגדנים עצמיים להערכת חומרת המחלה על כל חולה עם PAP. עד עכשיו, יכולת נטרול של GM-CSF נוגדנים עצמיים כבר העריכו ידי הערכת עיכוב הצמיחה של האדם תאים במח העצם או TF-1 תאים מגורה עם GM-CSF 6-8. במערכת המבדק, לעומת זאת, הוא לעתים קרובות בעייתי לקבל נתונים אמינים כמו גם להשוות את הנתונים ממעבדות שונות, בשל קשיים טכניים בשמירה על תאים במצב קבוע. המטרה: לחקות GM-CSF מחייב לקולטן GM-CSF על פני התא באמצעות קולטן תא-free-binding-assay. שיטות: טראנסגנטי תולעי משי הטכנולוגיה יושמה לקבלת כמות גדולה של מסיס רקומביננטי GM-CSF קולטני אלפא (sGMRα) עם הטוהר גבוהה 9-13. SGMRα רקומביננטי היה הכלולים שכבות sericin הידרופילי של חוטי משי מבלי התמזגו את החלבונים משי, ולכן, אנחנו יכולים בקלות לחלץ מתוך פקעות של טוהר טוב עם תמיסות מימיות נייטרלי 14,15. למרבה המזל, מבנים oligosaccharide, אשר הם קריטיים עבור מחייב עם GM-CSF, דומים יותר מבני האדם sGMRα מאלה המיוצרים על ידי חרקים או שמרים אחרים. תוצאות: מערכת תא ללא assay באמצעות sGMRα הניבו את הנתונים עם הפלסטיות אמינות גבוהה. GM-CSF מחייב sGMRα היה במינון dependently מעוכבים על ידי autoantibody polyclonal GM-CSF באופן דומה המבדק באמצעות TF-1 תאים, המציין כי חדש ללא התא שלנו assay המערכת באמצעות sGMRα שימושית יותר למדידת פעילות נטרול של GM-CSF נוגדנים עצמיים מאשר מערכת המבדק באמצעות TF-1 או תא אנושי לתאי מח עצם. מסקנות: הקמנו assay תא ללא לכימות יכולת נטרול של GM-CSF autoantibody.

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

עיצבנו assay תא ללא קולטן מחייב כדי לאמוד את הכריכה של גורם המושבה מגרה granulocyte-מקרופאג (GM-CSF) לקולטנים. זה מאפשר לנו להעריך עיכוב תחרותי של biotinylated GM-CSF מחייב מסיסים GM-CSF קולטן אלפא ידי GM-CSF autoantibody עם reproducibility מעולה.

Assay חינם Cell מערכת הערכת קיבולת נטרול של נוגדן GM-CSF באמצעות קולטן מסיס רקומביננטי GM-CSF

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

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Research

JoVE Journal

Immunology and Infection

12.6K Views.  Niigata University Medical and Dental Hospital. עיצבנו assay תא ללא קולטן מחייב כדי לאמוד את הכריכה של גורם המושבה מגרה granulocyte-מקרופאג (GM-CSF) לקולטנים. זה מאפשר לנו להעריך עיכוב תחרותי של biotinylated GM-CSF מחייב מסיסים GM-CSF קולטן אלפא ידי GM-CSF autoantibody עם reproducibility מעולה.

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.

Bioenergetics וחמצוני פרץ: הפרוטוקולים לבידוד ולהערכה של לויקוציטים אדם וטסיות הדם

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.

גבוהה Scalable תפוקה בחירה מספריות נוגדן סינטטיים-מוצג הפאג

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.

דור וזיהוי GM-CSF נגזר מכתשיים דמוי מאקרופאגים דנדריטים תאי עכבר מח עצם

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.

להערכת תפקוד רשתית microglial Phagocytic<em&gt; In vivo</em&gt; שימוש cytometry זרימה מבוסס Assay

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).

Murine לימפוציטים תיוג ידי<sup&gt; 64</sup&gt; Cu-נוגדן קולטן מיקוד עבור<em&gt; In vivo</em&gt; סחר Cell על ידי PET / CT

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.

דור של בריחה וריאציות של נטרול נוגדנים חד-שבטיים של וירוס שפעת

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.

מבחני מדידת שפעת בנטרול תגובות נוגדנים וירוסים A(H3N2) סרה האנושי על-ידי Microneutralization באמצעות תאים MDCK-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.

הרומן מזין ללא מערכת לייצור המוני של תאים רוצח טבעי מאתר במבחנה

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.

במבחנה הבידול של העכבר גורם גרנולוציט-מקרופאג-המושבה-מגרה (GM-CSF)-הפקת תאי T Helper (THGM)

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.