We thank Dr. Peter T. Sage for his assistance in generating some of the representative images. This work was supported by an NIH R01 grant to C.V.C. (HL104006).

<ol>
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The authors have nothing to disclose.

בסך הכל, פרוטוקול זה מתאר שיטות לחקירת תאי האנדותל כט) נגמ&quot;שים פיסיולוגיים understudied וii) כסוג חדש של &#39;מודל APC הסלולרי מישוריים&#39;. עם כל כבוד ללשעבר, זה הפך להיות יותר ויותר מוערך שנגמ&quot;שים שאינם hematopoietic ההיקפי (או &#39;סטרומה&#39;) ממלאים תפקידים קריטיים, שאינם מיותרים <em...

לימפוציטים מסוג T הוא סניף של המערכת החיסונית אדפטיבית המאופיינת ביכולת לזהות ביעילות אנטיגן פפטיד (Ag) חייבת מורכב histocompatibility גדול (MHC) מולקולות באמצעות קולטנים של תאי T (TCRs) 1. לימפוציטים נאיביים constitutively להעביר ולסרוק תאים מקצועיים Ag הצגה &#39;(נגמ&quot;שים; למשל, תאים דנדריטים) בתוך בלוטות הלימפה, בעוד תאי הזיכרון / T מפעיל צריכים לסקור מגוון רחב מאוד של נגמ&quot;שים ותאי יעד פוטנציאליים בתוך רקמות היקפי ביעילות. בדקות הבאות הכרה ראשונית של Ag מקור על APC, ימפוציטים לעצור הגירתם ומתחיל להיווצר ממשק תאי תאים אינטימי מיוחד &quot;סינפסה החיסונית &#39;כינתה (IS). ספג (כלומר, 30-60 דקות) היא קשר נדרש להגביר ולקיים איתות 2-7. מחקרים המתעוררים לזהות שבתוך הוא, זה הוא ההיווצרות רציפה ומהיר remodeling של מיקרו-אשכולות בדידים איתות תת-תאי (כלומר, מכילים MHC / Ag-TCR, F- אקטין, הדבקה ומולקולות איתות) הקובעים את החוזק והאיכות של תגובות חיסוני וכתוצאה מכך 2-7. עם זאת, פרטים דינמיים ומנגנון רגולציה של תהליך זה באופן חלקי הבינו 8,9. זה נובע במידה רבה מאתגרים טכניים הקשורים בטופולוגיות סדירות של משטחי APC ונטייה גרועים מבוקרת של מטוסי האינטראקציה תאי תאים, נושאים שעמוקים להגביל את ההדמיה spatiotemporal הנדרשת גישות 8-10 (Figure1A). <img alt="איור 1" src="/files/ftp_upload/53288/53288fig1.jpg" /> איור 1. דגם APC התא פיסיולוגי מישורי הדמיה החיסוני סינפסה Dynamics. סכמטי ממחיש הדמיה מסורתית של סינפסה החיסונית בין תאי T וprofessio<html> תא סופי APC () ו- T ומודל מסורתי שומנים מישוריים APC bilayer (ב &#39;) בהשוואה למודל זה רומן אנדותל APC מישוריים (C). נגמ&quot;שים מקצועיים לספק סינפסות החיסונית פיזיולוגית אבל מציעים ממשק מונחה גרוע תא-תא (כלומר, ביחס למישור ההדמיה XY האופטימלי; רזולוציה ~ 0.2 מיקרומטר), אשר באופן דרמטי פוגע מרחבית (רזולוציה ~ מיקרומטר מטוס ההדמיה z 1) ובזמן (כלומר, בשל הצורך לסרוק שוב ושוב בכל מטוסי ההדמיה z) רזולוציה של הדמיה. יש דגמי bilayer טופולוגיה מישוריים המספקת רזולוציה ההדמיה spatiotemporal אופטימלית, אבל גם פשוטים ביותר, שאינם פיסיולוגי ונוקשה. מודל תא האנדותל זה משלב את הטופולוגיה מישוריים של bilayers שומנים עם המצע הפיזיולוגי של APC קלאסי כדי לספק רזולוציה הדמיה מרחב ובזמן אופטימלית בסביבה פיזיולוגית.מ &#39;/ קבצים / ftp_upload / 53,288 53288fig1large.jpg &quot;target =&quot; / _ blank &quot;&gt; לחץ כאן כדי לצפות בגרסה גדולה יותר של דמות זו. העבודה קודמת עקפה באופן חלקי על המכשולים הללו על ידי פיתוח מודלים מצע מישוריים (כלומר, bilayers שומנים ומשטחים מצופים נוגדנים) המספקים רזולוציה spatiotemporal אופטימלית (כלומר, באמצעות תיקון משטח הפעלת תא T לתכנית אחת שמקבילה להדמית XY האופטימלית מטוס) 11-15 (1B איור). מודלים אלה הקלו תובנות חשובות דינמיקת subcellular / מולקולרית השולטות איתות אנטיגני בתאי T, כוללים גילוי אקטין דינמי / TCR מיקרו-אשכולות איתות 7,11-14. עם זאת, מודלים כאלה מטבעם פשטניים, כמו גם נוקשה (מניעת הפיתוח / המחקר של תכונות טופולוגי 3 ממדים) (איור 1). לכן, זה עדיין לא ברור איך להתייחס כגון ממצאים לPHYתאי תאים siologic מעקב חיסוני. למרות שעדיין understudied, כלי דם ותאי האנדותל הלימפה הם מתעוררים כגדולים (כלומר, גדולים יותר במספרים מכל נגמ&quot;שים המקצועיים, על ידי ~ 1000-פי) תא היקפי של נגמ&quot;שים &quot;חצי מקצועי&quot; 16-18. תאים אלה מבטאים MHC-אני-, MHC-II- ושפע של מולקולות שיתוף ממריץ (למשל, CD40, LFA3, ICOSL, 4-1BB, OX40L, TL1A, PD-L1, אבל לא CD80 וCD86) ואסטרטגיים ממוקם בממשק שבין דם לרקמה שבו הם משרתים פונקציות זקיף מיוחדות 16-18. מחקרים קודמים הראו שתאי האנדותל יכולים למעשה מחדש לעורר מפעיל / זיכרון, אבל לא נאיבי, תאי T 19-25. כך, תאי האנדותל עשויים לשחק תפקידי APC ייחודיים בשלב מפעיל של תגובות חיסוניים אדפטיבית בתוך הרקמות ההיקפית, כגון השפעה מקומית על הפעלת תא T, בידול, זיכרון וסובלנות 16,17,26. CRItically, כאשר גדלו במבחנה, תאי האנדותל יוצרים משטחי תא כמעט מישורי והם בקלות transfectable (למשל, עם כתבי חלבון פלואורסצנטי). תכונות אלה הן אידיאליים עבור הדמיה גבוהה spatiotemporal רזולוציה של דינמיקה טופולוגית במהלך אינטראקציות תא-תא 19,27. כך תאי האנדותל עשויים לשמש כמודל &#39;מישוריים סלולארי APC&#39; פיסיולוגי מובהק מתאים ללימוד מנגנוני שיפוץ subcellular / מולקולריים המניעה את הכרת אנטיגן ולווסת תגובות (איור 1 ג) 19,20. בעבר הוקם טכניקות הדמיה משלימות (כולל transfection של תאי endothelia עם מקבלי חלבון פלואורסצנטי של קרום הפלזמה וcytosol) ללומד את הפרטים של אינטראקציה ליקוציט-האנדותל בהידבקות וההגירה Transendothelial 27, הראה כי לויקוציטים באופן פעיל לחקור את פני השטח של האנדותל ידי דינמי הכנסהההכחשה ד של תת-מיקרון בקנה מידה, בליטות גליליות אקטין עשיר (~ 200-1,000 ננומטר בקוטר ועומק) כינו בליטות כמו invadosome (כלומר, &#39;ILPs&#39;) 27,28. הדמיה גישות אלה הורחבו עוד יותר יחד עם יצירת הפרוטוקולים לנצל פונקצית APC אנדותל לפתח השיטות הראשונות להדמיה גבוהה spatiotemporal רזולוציה של סינפסה החיסונית תא אנדותל T כ19,20 דיווח ועוד לתאר זאת. ממצא מרכזי הנגזר ממודל APC סלולארי זה מישוריים רומן הוא שILPs תא T לתפקד גם בקידום זיהוי Ag ראשוני ובשמירת איתות שלאחר מכן. ואכן, מערכים של ILPs מרובה (שהתייצבו ונצבר בתגובה הראשונית שטף סידן) להעשרת תכנית בTCR ומולקולות רמיזות של PKC-Q איתות פעילה כגון, ZAP-70, phosphotyrosine וHS1. לכן, ILPs נראה לייצג מקביל פיסיולוגי תלת-ממדית למייקרו-TCR האיתותאשכולות ראו בדגמי bilayer מישוריים. גישה זו, ובכך, ברגישות מגלה / דינמיקה מולקולרית ואדריכלית (ומשתמע ביו-מכאנית) דיווחים לא אחרת לזיהוי. השיטה המתוארת במסמך זה צריכה להיות שימושי לגישור על הפער בין APC המקצועי ודגמי מצע APC מלאכותיים כדי לשפר את היכולת שלנו לחקור את המנגנונים בסיסיים של תגובות חיסוניים אדפטיבית. ואילו כאן הדגש הוא על ההפעלה של מפעיל CD4 + סוג Th1 / תא זיכרון, גישה בסיסית זו ניתן לשנות בקלות ללמוד מגוון רחב של סוגי תאי T וAGS, כמפורט להלן.

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		<tr height="21">
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			<td style="width:152px;">13-678-8D</td>
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			<td height="21" style="height:21px;width:392px;">&#160;ICAM1 mouse anti-human</td>
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			<td height="21" style="height:21px;">HS1 mouse anti-human</td>
			<td>BD Biosciences</td>
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			<td height="21" style="height:21px;">Anti-Human CD11a (LFA-1alpha) Purified</td>
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			<td>ebioscience</td>
			<td>53-0037-41</td>
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			<td height="21" style="height:21px;">Anti-MHC Class II antibody&#160;</td>
			<td style="width:149px;">Abcam</td>
			<td>ab55152</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Anti-Talin 1 antibody</td>
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			<td height="21" style="height:21px;width:392px;">Anti-PKC theta antibody&#160;</td>
			<td style="width:149px;">Abcam</td>
			<td>ab109481</td>
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			<td height="21" style="height:21px;">phosphotyrosine (4G10 Platinum)</td>
			<td style="width:149px;">Millipore</td>
			<td>50-171-463</td>
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			<td>Required electroporator for step 4</td>
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			<td height="41" style="height:41px;width:392px;">Zeiss Axiovert</td>
			<td style="width:149px;">Carl Zeiss MicroImaging</td>
			<td style="width:152px;"></td>
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		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:392px;">Zeiss LSM510&#160;</td>
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			<td></td>
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		<tr height="41">
			<td height="41" style="height:41px;width:392px;">Zeiss Axiovison Software</td>
			<td style="width:149px;">Carl Zeiss MicroImaging</td>
			<td style="width:152px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">NU-425 (Series 60) Biological Safety Cabinet</td>
			<td style="width:149px;">NuAIRE</td>
			<td style="width:152px;">Nu-425-600</td>
			<td></td>
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		<tr height="41">
			<td height="41" style="height:41px;width:392px;">&#160;Forma STRCYCLE 37 &#176;C, 5% CO2 Cell culture Incubator</td>
			<td>Fisher Scientific</td>
			<td>202370</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:392px;">Centrifuge 5810</td>
			<td style="width:149px;">Eppendorf</td>
			<td style="width:152px;">EW-02570-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">Hemocytometer</td>
			<td style="width:149px;">Sigma-Aldrich</td>
			<td style="width:152px;">&#160;Z359629</td>
			<td>Bright-Line Hemocytometer</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:392px;">Isotemp Waterbath model 202</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td style="width:152px;">15-462-2</td>
			<td></td>
		</tr>
	</tbody>
</table>

an endothelial planar cell model for imaging immunological synapse dynamics

חסינות מסתגלת מוסדרת על ידי אינטראקציות דינמיות בין תאי T ותאי הצגת אנטיגן (&#39;נגמ&quot;שים&#39;) המכונה &quot;סינפסות החיסונית&quot;. בתוך ממשקי תאי תאים האינטימיים אלה אשכולות בדידים משנה הסלולר של MHC / Ag-TCR, F- אקטין, הדבקה ויוצרים מולקולות איתות ולשפץ במהירות. דינמיקה אלה נחשבות לגורמים קריטיים של שתי היעילות והאיכות של התגובות חיסוני המתפתחות ולכן של מגן לעומת חסינות פתולוגיים. הבנה נוכחית של סינפסות החיסונית עם נגמ&quot;שים פיסיולוגיים היא מוגבלת על ידי חוסר ההתאמה של הרזולוציה ההדמיה השגה. למרות שמודלים מלאכותיים מצע (למשל, bilayers שומנים מישוריים) מציעים רזולוציה מעולה והיו כלים יקרים מאוד, הם מטבעם אינו פיסיולוגיים ופשטניים. תאי האנדותל של כלי דם והלימפה צמחו כתא רקמה היקפית חשובה (או סטרומה) ​​של &#39;חצי מקצועאל נגמ&quot;שים &quot;. נגמ&quot;שים אלה (המבטאים ביותר של המכונות מולקולריות של נגמ&quot;שים מקצועיים) יש התכונה הייחודית של יצירת תא שטח כמעט מישורי והם בקלות transfectable (למשל, עם כתבי חלבון פלואורסצנטי). במסמך גישה בסיסית ליישום תאי האנדותל כרומן ו&#39;מודל APC הסלולרי מישוריים &#39;פיסיולוגי להדמיה וחקירה של תהליכי איתות אנטיגני הבסיסיים משופרות תתואר.

גישת הדמיה רומן באמצעות תאי האנדותל ושילוב יתרונות הרזולוציה של מודל bilayers שומנים מישוריים עם המורכבות וdeformability של נגמ&quot;שים מקצועיים הפיזיולוגיים פותחה (איור 1). איור 2 מספק דוגמאות של הגירה טיפוסית, שטף סידן ודינמיקה טופולוגית נצ...

Watch this Scientific Journal Video about An Endothelial Planar Cell Model for Imaging Immunological Synapse Dynamics at JoVE.com

An Endothelial Planar Cell Model for Imaging Immunological Synapse Dynamics

Adaptive immunity is controlled by dynamic 'immunological synapses' formed between T cells and antigen presenting cells. This protocol describes methods for investigating endothelial cells both as understudied physiologic APCs and as a novel type of 'planar cellular APC model'.

דגם תא האנדותל מישורי הדמיה החיסוני סינפסה Dynamics

Adaptive immunity is regulated by dynamic interactions between T cells and antigen presenting cells (&#39;APCs&#39;) referred to as &#39;immunological ...

an-endothelial-planar-cell-model-for-imaging-immunological-synapse

Research

JoVE Journal

Immunology and Infection

8.8K Views.  Harvard Medical School - BIDMC. Adaptive immunity is controlled by dynamic 'immunological synapses' formed between T cells and antigen presenting cells. This protocol describes methods for investigating endothelial cells both as understudied physiologic APCs and as a novel type of 'planar cellular APC model'.

Video: An Endothelial Planar Cell Model for Imaging Immunological Synapse Dynamics

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between CD4+ T cells involves the formation of a virological synapse (VS), which is an F-actin-dependent cell-cell junction formed upon the engagement of HIV-1 envelope gp120 on the infected cell with CD4 and the chemokine receptor (CKR) CCR5 or CXCR4 on the target cell 8. In addition to gp120 and its receptors, other membrane proteins, particularly the adhesion molecule LFA-1 and its ligands, the ICAM family, play a major role in VS formation and virus transmission as they are present on the surface of virus-infected donor cells and target cells, as well as on the envelope of HIV-1 virions1,4,5,6,7,13. VS formation is also accompanied by intracellular signaling events that are transduced as a result of gp120-engagement of its receptors. Indeed, we have recently showed that CD4+ T cell interaction with gp120 induces recruitment and phosphorylation of signaling molecules associated with the TCR signalosome including Lck, CD3&zeta;, ZAP70, LAT, SLP-76, Itk, and PLC&gamma;15.
In this article, we present a method to visualize supramolecular arrangement and membrane-proximal signaling events taking place during VS formation. We take advantage of the glass-supported planar bi-layer system as a reductionist model to represent the surface of HIV-infected cells bearing the viral envelope gp120 and the cellular adhesion molecule ICAM-1. The protocol describes general procedures for monitoring HIV-1 gp120-induced VS assembly and signal activation events that include i) bi-layer preparation and assembly in a flow cell, ii) injection of cells and immunofluorescence staining to detect intracellular signaling molecules on cells interacting with HIV-1 gp120 and ICAM-1 on bi-layers, iii) image acquisition by TIRF microscopy, and iv) data analysis. This system generates high-resolution images of VS interface beyond that achieved with the conventional cell-cell system as it allows detection of distinct clusters of individual molecular components of VS along with specific signaling molecules recruited to these sub-domains.

This article describes a method to visualize formation of an HIV-1 envelope-induced virological synapse on glass supported planar bilayers by total internal reflection fluorescence (TIRF) microscopy. The method can also be combined with immunofluorescence staining to detect activation and redistribution of signaling molecules that occur during HIV-1 envelope-induced virological synapse formation.

הדמיה של HIV-1 מעטפה הנגרמת סינפסה Virological והאיתות על bilayers שומנים סינתטי

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between ...

Visualization of the Immunological Synapse by Dual Color Time-gated Stimulated Emission Depletion (STED) Nanoscopy

Natural killer cells form tightly regulated, finely tuned immunological synapses (IS) in order to lyse virally infected or tumorigenic cells. Dynamic actin reorganization is critical to the function of NK cells and the formation of the IS. Imaging of F-actin at the synapse has traditionally utilized confocal microscopy, however the diffraction limit of light restricts resolution of fluorescence microscopy, including confocal, to approximately 200 nm. Recent advances in imaging technology have enabled the development of subdiffraction limited super-resolution imaging. In order to visualize F-actin architecture at the IS we recapitulate the NK cell cytotoxic synapse by adhering NK cells to activating receptor on glass. We then image proteins of interest using two-color stimulated emission depletion microscopy (STED). This results in &#60;80 nm resolution at the synapse. Herein we describe the steps of sample preparation and the acquisition of images using dual color STED nanoscopy to visualize F-actin at the NK IS. We also illustrate optimization of sample acquisition using Leica SP8 software and time-gated STED. Finally, we utilize Huygens software for post-processing deconvolution of images.

Visualization of the Immunological Synapse by Dual Color Time-gated Stimulated Emission Depletion STED Nanoscopy

Here we illustrate the protocol for imaging by two-color STED nanoscopy the cytotoxic immune synapse of NK cells recapitulated on glass. Using this method we obtain sub-100 nm resolution of synapse proteins and the cytoskeleton.

ויזואליזציה של סינפסה החיסונית על ידי פליטה מאולצת דלדול מגודרת בזמן צבע כפול (STED) Nanoscopy

Natural killer cells form tightly regulated, finely tuned immunological synapses (IS) in order to lyse virally infected or tumorigenic cells. Dynamic ...

Real-time Imaging of Endothelial Cell-cell Junctions During Neutrophil Transmigration Under Physiological Flow

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known as leukocyte transendothelial migration. Two routes for leukocytes to cross the endothelial monolayer have been described: the paracellular route, i.e., through the cell-cell junctions and the transcellular route, i.e., through the endothelial cell body. However, it has been technically difficult to discriminate between the para- and transcellular route. We developed a simple in vitro assay to study the distribution of endogenous VE-cadherin and PECAM-1 during neutrophil transendothelial migration under physiological flow conditions. Prior to neutrophil perfusion, endothelial cells were briefly treated with fluorescently-labeled antibodies against VE-cadherin and PECAM-1. These antibodies did not interfere with the function of both proteins, as was determined by electrical cell-substrate impedance sensing and FRAP measurements. Using this assay, we were able to follow the distribution of endogenous VE-cadherin and PECAM-1 during transendothelial migration under flow conditions and discriminate between the para- and transcellular migration routes of the leukocytes across the endothelium.

Leukocytes cross the endothelial monolayer using the paracellular or the transcellular route. We developed a simple assay to follow the distribution of endogenous junctional VE-cadherin and PECAM-1 during leukocyte transendothelial migration under physiological flow to discriminate between the two transmigration routes.

הדמיה בזמן אמת של תאי תאי צומת האנדותל במהלך נויטרופילים גלגול תחת פיזיולוגית זרימה

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known ...

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter lesions in inflammatory demyelinating disorders like multiple sclerosis (MS). In these disorders, mainly CD8+ T-cells of putative specificity for myelin- and oligodendrocyte-related antigens are found, so that neuronal apoptosis in grey matter lesions may be a collateral effect of these cells. Different types of animal models are established to study the underlying mechanisms of the mentioned pathophysiological processes. However, although they mimic some aspects of MS, it is impossible to dissect the exact mechanism and time course of &#8216;&#8216;collateral&#8217;&#8217; neuronal cell death. To address this course, here we show a protocol to study the mechanisms and time response of neuronal damage following an oligodendrocyte-directed CD8+ T cell attack. To target only the myelin sheath and the oligodendrocytes, in vitro activated oligodendrocyte-specific CD8+ T-cells are transferred into acutely isolated brain slices. After a defined incubation period, myelin and neuronal damage can be analysed in different regions of interest. Potential applications and limitations of this model will be discussed.

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

To address mechanisms of demyelination and neuronal apoptosis in cortical lesions of inflammatory demyelinating disorders, different animal models are used. We here describe an ex vivo approach by using oligodendrocyte-specific CD8+ T-cells on brain slices, resulting in oligodendroglial and neuronal death. Potential applications and limitations of the model are discussed.

<em&gt; Ex vivo</em&gt; דגם של התקפת T-Cell-מכוון oligodendrocyte בפרוסות המוח חריף

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter ...

Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has been in the study of immunological synapse formation, due to the ability of the glass-supported planar lipid bilayers to mimic the surface of a target cell while forming a horizontal interface. The recent advances in super-resolution imaging have further allowed scientists to better view the fine details of synapse structure. In this study, one of these advanced techniques, stimulated emission depletion (STED), is utilized to study the structure of natural killer (NK) cell synapses on the supported lipid bilayer. Provided herein is an easy-to-follow protocol detailing: how to prepare raw synthetic phospholipids for use in synthesizing glass-supported bilayers; how to determine how densely protein of a given concentration occupies the bilayer's attachment sites; how to construct a supported lipid bilayer containing antibodies against NK cell activating receptor CD16; and finally, how to image human NK cells on this bilayer using STED super-resolution microscopy, with a focus on distribution of perforin positive lytic granules and filamentous actin at NK synapses. Thus, combining the glass-supported planar lipid bilayer system with STED technique, we demonstrate the feasibility and application of this combined technique, as well as intracellular structures at NK immunological synapse with super-resolution.

We describe here a combination of the glass-supported lipid bilayer technique of forming immunological synapses with the super-resolution imaging technique of stimulated emission depletion (STED) microscopy. The goal of this protocol is to provide users with the instructions necessary to successfully carry out these two techniques.

הדמיה ברזולוציה סופר של תא הרוצח הטבעי החיסונית סינפסה על מישורי שומנים bilayer נתמך-זכוכית

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has ...

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). This process is characterized by the transmigration of activated T cells across brain endothelial cells (ECs), the main constituents of the BBB. However, the consequences on brain EC function upon interaction with such T cells are largely unknown. Here we describe an assay that allows for the evaluation of primary mouse brain microvascular EC (MBMEC) function and barrier integrity during the interaction with T cells over time. The assay makes use of impedance cell spectroscopy, a powerful tool for studying EC monolayer integrity and permeability, by measuring changes in transendothelial electrical resistance (TEER) and cell layer capacitance (Ccl). In direct contact with ECs, stimulated but not na&#239;ve T cells are capable of inducing EC monolayer dysfunction, as visualized by a decrease in TEER and an increase in Ccl. The assay records changes in EC monolayer integrity in a continuous and automated fashion. It is sensitive enough to distinguish between different strengths of stimuli and levels of T cell activation and it enables the investigation of the consequences of a targeted modulation of T cell-EC interaction using a wide range of substances such as antibodies, pharmacological reagents and cytokines. The technique can also be used as a quality control for EC integrity in in vitro T-cell transmigration assays. These applications make it a versatile tool for studying BBB properties under physiological and pathophysiological conditions.

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Here, we describe an in vitro murine model of the blood-brain barrier that makes use of impedance cell spectroscopy, with a focus on the consequences on endothelial cell integrity and permeability upon interaction with activated T cells.

<em&gt; במבחנה</em&gt; דגם של מחסום דם-מוח באמצעות ספקטרוסקופיית עכבה: התמקדות T Cell-אנדותל אינטראקציה תא

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal ...

Studying Organelle Dynamics in B Cells During Immune Synapse Formation

Recognition of surface-tethered antigens&#160;by the B cell receptor (BCR)&#160;triggers the formation of an immune synapse (IS), where both signaling&#160;and antigen uptake are coordinated. IS formation involves dynamic actin remodeling accompanied by the polarized recruitment to the synaptic membrane of the centrosome and associated intracellular organelles such as lysosomes and the Golgi apparatus. Initial stages of actin remodeling allow B cells to increase their cell surface and maximize the quantity of antigen-BCR complexes gathered at the synapse. Under certain conditions, when B cells recognize antigens associated to rigid surfaces, this process is coupled to the local recruitment and secretion of lysosomes, which can facilitate antigen extraction. Uptaken antigens&#160;are internalized into specialized endo-lysosome compartments for processing into peptides, which are loaded onto major histocompatibility complex II (MHC-II) molecules for further presentation to T helper cells. Therefore, studying organelle dynamics associated with the formation of an IS is crucial to understanding how B cells are activated. In the present article we will discuss both imaging and a biochemical technique used to study changes in intracellular organelle positioning and cytoskeleton rearrangements that are associated with the formation of an IS in B cells.

Herein we describe two approaches to characterize cell polarization events in B lymphocytes during the formation of an IS. The first, involves quantification of organelle recruitment and cytoskeleton rearrangements at the synaptic membrane. The second is a biochemical approach, to characterize changes in composition of the centrosome, which undergoes polarization to the immune synapse.

לומדת אורגל דינמיקה ב-B תאים במהלך היווצרות סינפסה החיסונית

Recognition of surface-tethered antigens&#160;by the B cell receptor (BCR)&#160;triggers the formation of an immune synapse (IS), where both ...

Implantation and Monitoring by PET/CT of an Orthotopic Model of Human Pleural Mesothelioma in Athymic Mice

Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor arising in the mesothelium that covers the lungs, the heart, and the thoracic cavity. MPM development is mainly associated with asbestos. Treatments provide only modest survival since the median survival average is 9&#8211;18 months from the time of diagnosis. Therefore, more effective treatments must be identified. Most data describing new therapeutic targets were obtained from in vitro experiments and need to be validated in reliable in vivo preclinical models. This article describes one such reliable MPM orthotopic model obtained after injection of a human MPM cell line H2052/484 into the pleural cavity of immunodeficient athymic mice. Transplantation in the orthotopic site allows studying the progression of tumor in the natural in vivo environment. Positron emission tomography/computed tomography (PET/CT) molecular imaging using the clinical [18F]-2-fluoro-2-deoxy-D-glucose ([18F]FDG) radiotracer is the diagnosis method of choice for examining patients with MPM. Accordingly, [18F]FDG-PET/CT was used to longitudinally monitor the disease progression of the H2052/484 orthotopic model. This technique has a high 3R potential (Reduce the number of animals, Refine to lessen pain and discomfort, and Replace animal experimentation with alternatives) since the tumor development can be monitored non-invasively and the number of animals required could be significantly reduced.
This model displays a high development rate, a rapid tumor growth, is cost-efficient and allows for rapid clinical translation. By using this orthotopic xenograft MPM model, researchers can assess biological responses of a reliable MPM model following therapeutic interventions.

This article describes the generation of an orthotopic mouse model of human pleural mesothelioma by implantation of H2052/484 mesothelioma cells into the pleural cavity of immunocompromised athymic mice. The longitudinal monitoring of the development of intrapleural tumors was assessed by non-invasive multimodal [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography and computed tomography imaging.

השרשה וניטור על ידי PET/CT של מודל אורתוטופית של מזותליומה של האדם בעכברים האטורי

Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor arising in the mesothelium that covers the lungs, the heart, and the thoracic cavity. ...

Imaging the Human Immunological Synapse

The purpose of the method is to generate an immunological synapse (IS), an example of cell-to-cell conjugation formed by an antigen-presenting cell (APC) and an effector helper T lymphocyte (Th) cell, and to record the images corresponding to the first stages of the IS formation and the subsequent trafficking events (occurring both in the APC and in the Th cell). These events will eventually lead to polarized secretion at the IS. In this protocol, Jurkat cells challenged with Staphylococcus enterotoxin E (SEE)-pulsed Raji cells as a cell synapse model was used, because of the closeness of this experimental system to the biological reality (Th cell-APC synaptic conjugates). The approach presented here involves cell-to-cell conjugation, time-lapse acquisition, wide-field fluorescence microscopy (WFFM) followed by image processing (post-acquisition deconvolution). This improves the signal-to-noise ratio (SNR) of the images, enhances the temporal resolution, allows the synchronized acquisition of several fluorochromes in emerging synaptic conjugates and decreases fluorescence bleaching. In addition, the protocol is well matched with the end point cell fixation protocols (paraformaldehyde, acetone or methanol), which would allow further immunofluorescence staining and analyses. This protocol is also compatible with laser scanning confocal microscopy (LSCM) and other state-of-the-art microscopy techniques. As a main caveat, only those T cell-APC boundaries (called IS interfaces) that were at the right 90&#176; angle to the focus plane along the Z-axis could be properly imaged and analyzed. Other experimental models exist that simplify imaging in the Z dimension and the following image analyses, but these approaches do not emulate the complex, irregular surface of an APC, and may promote non-physiological interactions in the IS. Thus, the experimental approach used here is suitable to reproduce and to confront some biological complexities occurring at the IS.

This protocol images both the immunological synapse formation and the subsequent polarized secretory traffic towards the immunological synapse. Cellular conjugates were formed between a superantigen-pulsed Raji cell (acting as an antigen-presenting cell) and a Jurkat clone (acting as an effector helper T lymphocyte).

הדמיה של הסינפסה החיסונית של האדם

The purpose of the method is to generate an immunological synapse (IS), an example of cell-to-cell conjugation formed by an antigen-presenting cell (APC) ...

An Immunological Model for Heterotopic Heart and Cardiac Muscle Cell Transplantation in Rats

Heterotopic heart transplantation in rats has been a commonly used model for diverse immunological studies for more than 50 years. Several modifications have been reported since the first description in 1964. After 30 years of performing heterotopic heart transplantation in rats, we have developed a simplified surgical approach, which can be easily taught and performed without further surgical training or background.
After dissection of the ascending aorta and the pulmonary artery and ligation of superior and inferior caval and pulmonary veins, the donor heart is harvested and subsequently perfused with ice-cold saline solution supplemented with heparin. After clamping and incising the recipient abdominal vessels, the donor ascending aorta and pulmonary artery are anastomosed to the recipient abdominal aorta and inferior vena cava, respectively, using continuous running sutures.
Depending on different donor-recipient combinations, this model allows analyses of either acute or chronic rejection of allografts. The immunological significance of this model is further enhanced by a novel approach of in-ear injection of vital cardiac muscle cells and subsequent analysis of draining cervical lymphatic tissue.

We describe a model of heterotopic abdominal heart transplantation in rats, implying modifications of current strategies, which lead to a simplified surgical approach. Additionally, we describe a novel rejection model by in-ear injection of vital cardiac muscle cells, allowing further transplant immunological analyses in rats.

מודל אימונוולוגי עבור הלב הטרוולפית ושריר הלב השתלת תא בחולדות

Heterotopic heart transplantation in rats has been a commonly used model for diverse immunological studies for more than 50 years. Several modifications ...