D.M.O. acknowledges European Research Council grant No. 337187 and the Marie-Curie Career Integration Grant No. 334303. P.W.W. acknowledges the Natural Sciences and Engineering Research Council of Canada's Discovery Grant program.

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The authors declare that they have no competing financial interests.

טכניקה זו מאפשרת לחוקרי תמונה ולכמת בעבר פרטים פתירים בתאים לבטא הקרינה. בתוצאות שלנו מראים כי F- אקטין זורם בצורה סימטרית מהפריפריה התא לכיוון מרכז התא בתא T הוא. הממצאים אלה עולים בקנה אחד עם תוצאות קודמות מנתוני רשם גלים פרופיל קו 1D 13 ולהאריך את היכולת של ניתו?...

מטרת הניסוי היא תמונה ומאפיין את הזרימה המולקולרית של filamentous- אקטין (F-) בתאים חיים T, שחורגת ממגבלת העקיפה כדי להקים זרימת כיוון ועצמה במהלך סינפסה החיסונית (IS) היווצרות. טכניקה זו תבוצע באמצעות מיקרוסקופ מובנה תאורה (SIM) במצב הקרינה השתקפות פנימית (TIRF) כולל, הדמיה Jurkat תאי T יוצרים סינפסה מלאכותית על הפעלת coverslips מצופה אנטי-CD3- ונוגדנים נגד CD28. הצורות היא בין תא T והיעד שלה; תא הצגת אנטיגן (APC) על קולטן תא T 1,2 הפעלה (TCR). כמו זה הוא הצעד הראשון בקביעה האם המערכת החיסונית אדפטיבית יוצרת תגובה חיסונית לזיהום, את ההשלכות של היענות לגירויים נכונה יכולות לגרום למספר המחלות. החשיבות של האינטראקציה התא T-APC מתועדת היטב, לעומת זאת, התפקיד (ים) של הבוגר הוא לאחר סי TCR הראשוניתהפנמת gnal הם עדיין לא הבינו באופן מלא. כחשב שלד התא כדי לסייע שני תהליכים הקשורים להפעלת TCR (התנועה של מולקולות בקרום הפלזמה והשליטה של מולקולות איתות תלויות ביקטין שלד תא 3,4), הבנה כיצד שלד התא מארגן מחדש מרחב ובזמן יבהיר את הצעדים של ארגון מחדש מולקולרי במהלך היווצרות סינפסה. הוא חשב זרימת מדרדר של F- אקטין לכלוא אשכולות TCR לכיוון מרכז סינפסה החיסונית, הוא האמין טרנסלוקציה זה להיות חיוני להפסקת אות ומחזור; סיוע לאיזון העדין של הפעלת תא T 5. בעוד מיקרוסקופ פלואורסצנטי הסטנדרטי הוא כלי גמיש שממשיך לספק תובנה על אירועים ביולוגיים רבים, כולל אינטראקציות תא-תא, הוא מוגבל על ידי היכולת של המערכת כדי לפתור במדויק fluorophores מרובה המתגורר קרוב יותר מdiffrמגבלת פעולה (≈200 ננומטר) של אחד את השני. אירועים מולקולריים רבים בתוך תאים כרוכים אוכלוסיות צפופות של מולקולות ולהציג סידור דינמי או בקפאון או על גירוי מסוים, מיקרוסקופ אור הרגילה אינה מספקת את התמונה המלאה. השימוש בהדמיה ברזולוציה סופר אפשר לחוקרים לעקוף את מגבלת העקיפה תוך שימוש במגוון של טכניקות. מיקרוסקופיה לוקליזציה מולקולה בודדת (SMLM) כגון PALM ו6,7 STORM יש את היכולת לפתור את המיקום של מולקולות עד דיוק של כ -20 ננומטר, לעומת זאת, טכניקות אלה מסתמכים על פי רכישה ארוכה, בניית תמונה על מסגרות רבות . בדרך כלל זה דורש דגימות להיות קבועים או למבנים להציג ניידות נמוכה fluorophores כך בודדת לא כהלכה כמספר רב של נקודות. בעוד דלדול פליטה מאולץ (STED) הדמיה מאפשרת רזולוציה סופר דרך עירור confocal מאוד סלקטיבית 8, הסריקה הנדרשתגישה יכולה להיות איטית מעל שדות תא כל מבט. STED גם מדגים phototoxicity המשמעותי לרזולוציות גבוהות יותר כתוצאה מהעלייה בכוחה של קרן הדלדול. מיקרוסקופיה תאורה המובנה (SIM 9) מספקת אלטרנטיבה לשיטות אלה, מסוגלים להכפיל את הרזולוציה של מיקרוסקופ פלואורסצנטי סטנדרטי והוא תואם יותר עם ​​הדמיה תא חי מטכניקות SMLM הנוכחיות. היא משתמשת בסמכויות לייזר נמוכות, במערכת רחבה בתחום לשדות כל תא מבט; מיועד לספק מוגבר במהירויות רכישה, והוא תואם עם תיוג fluorophore סטנדרטי. הטבע רחב בתחום של SIM גם מאפשר הניצול של הקרינה הכוללת הפנימית השתקפות (TIRF) לעירור סלקטיבית, עם עומק חדירה בממד הצירי של &lt;100 ננומטר. ספקטרוסקופיה מתאם תמונה (ICS) היא שיטה של ​​correlating תנודות בעוצמת הקרינה. אבולוציה של ICS; im מרחב ובזמןספקטרוסקופיה מתאם גיל (מאפיינים אילו 10) קורלציה אותות ניאון תאיים בזמן ובמרחב. באמצעות התאמת עוצמות פיקסל עם סובבי פיקסלים במסגרות מפגרת באמצעות פונקציית מתאם, מידע צבר לגבי זרימת מהירויות ויווניות. כדי להבטיח אובייקטים סטטיים לא להפריע לניתוח המאפיינים אילו, מסנן אובייקט נייח מיושם, אשר עובד על ידי הפחתת ממוצע נע של עוצמות פיקסל. הוא האמין הטכניקה המוצגת כאן להיות ההפגנה הראשונה של ספקטרוסקופיה מתאם תמונה מיושמת לנתונים במיקרוסקופ ברזולוציה הסופר לכמת את הזרימה המולקולרית בתאים חיים 11. שיטה זו משפרת בהדמיה עקיפה המוגבל של זרימת F- אקטין באמצעות ניתוח המאפיינים אילו 12 ותתאים חוקרים שרוצים לקבוע זרימה מולקולרית דו-ממדית בתאים חיים, מנתונים שנרכשו ברזולוציות מרחבי שמעבר לגבול השתברות אור.

<table border="0" cellpadding="0" cellspacing="0" width="799">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:271px;">Consumables:</td>
			<td style="width:245px;"></td>
			<td style="width:123px;"></td>
			<td style="width:160px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Jurkat E6.1 cells</td>
			<td>APCC</td>
			<td>ATTC TIB-152</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RPMI</td>
			<td>Life Technologies</td>
			<td>21875-091</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FBS</td>
			<td>Sigma</td>
			<td>F7524-500ML</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin-Strepromycin&#160;</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HBSS</td>
			<td>&#160;Life Technologies</td>
			<td>14025-050</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES</td>
			<td>Life Technologies</td>
			<td>15630-056</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">#1.5 8-well Labtek coverslips</td>
			<td>NUNC, Labtek</td>
			<td>155409</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LifeAct GFP construct</td>
			<td>Ibidi</td>
			<td>60101</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">OptiMem</td>
			<td>Life Technologies</td>
			<td>51985-042</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-CD3</td>
			<td>Cambridge Bioscience</td>
			<td>317315</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-CD28</td>
			<td>BD Bioscience / Insight Biotechnology</td>
			<td>555725</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS</td>
			<td>Life Technologies</td>
			<td>20012-019</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lens oil</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Name</td>
			<td>Company</td>
			<td>Catalog number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Equipment:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gene Pulsar Xcell System</td>
			<td>BioRad</td>
			<td>165-2660</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cuvette (0.4cm)</td>
			<td>BioRad</td>
			<td>165-2088</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Centrifuge (5810R)</td>
			<td>Eppendorf&#160;</td>
			<td>5805 000.327</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Centrifuge (Heraeus)</td>
			<td>Biofuge pico</td>
			<td>75003235</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">6 well plate</td>
			<td>Corning</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Stage-top incubator</td>
			<td>Tokai Hit</td>
			<td>INUH-TIZSH</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nikon N-SIM microscope</td>
			<td>Nikon</td>
			<td>Contact company</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nikon Analyse software</td>
			<td>Nikon</td>
			<td>Contact company</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Incubator (190D)</td>
			<td>LEEC</td>
			<td>Contact company</td>
			<td></td>
		</tr>
	</tbody>
</table>

cortical actin flow in t cells quantified by spatio-temporal image correlation spectroscopy of structured illumination microscopy data

פילמנטיות אקטין ממלא תפקיד מכריע ברוב התהליכים בתא כוללים תנועתיות ו, בתאי מערכת חיסון, ההיווצרות של אינטראקציה תאי תאי מפתח הידועה כסינפסה החיסונית. F- אקטין גם העריך לשחק תפקיד בויסות החלוקה מולקולרית בקרום של תאים כוללים דינמיקת שלפוחית ​​תת-קרומי ואשכולות חלבון. בעוד טכניקות מיקרוסקופ האור סטנדרטית מאפשרות תצפיות כלליות ועקיפות מוגבלים להתבצע, אירועים תאיים ומולקולריים רבים, כולל אשכולות וזרימה מולקולרית מתרחשים באוכלוסיות באורך-מאזניים הרבה מתחת לכח בפתרון של מיקרוסקופ אור הרגילה. על ידי שילוב כוללת הקרינה השתקפות הפנימית עם מיקרוסקופיה תאורת סופר ההדמיה ברזולוציה השיטה מובנה, הזרימה המולקולרית דו-ממדית של F- אקטין בסינפסה החיסונית של תאי T נרשמה. ספקטרוסקופיה מרחב ובזמן קורלציה תמונה (מאפיינים אילו) ולאחר מכן יושם, אשר מייצרת resul כימותTS בצורה של היסטוגרמות מהירות ומפות וקטור המייצגות זרימת יווניות וגודל. פרוטוקול זה מתאר את השילוב של טכניקות הדמיה ומאפיינים אילו ברזולוציה הסופר לייצר תזרים וקטורים ברמות תת-עקיפה של פרט. טכניקה זו שימשה כדי לאשר זרימה אקטין שהיא סימטרי מדרדר והצנטריפטלי ברחבי הפריפריה של תאי T על היווצרות סינפסה.

הגדרות מיקרוסקופ לאחר הצלחת השגת כל צעדים החוקר יש לי הדמיה מושלמת תאורה מובנה (SIM) של זרימת F- אקטין בסינפסה תא T (וידאו 1), ולכמת את הזרימה מולקולרית זה על ידי ספקטרוסקופיה מרחב ובזמן קורלציה תמו?...

Watch this Scientific Journal Video about Cortical Actin Flow in T Cells Quantified by Spatio-temporal Image Correlation Spectroscopy of Structured Illumination Microscopy Data at JoVE.com

Cortical Actin Flow in T Cells Quantified by Spatio-temporal Image Correlation Spectroscopy of Structured Illumination Microscopy Data

To investigate flow velocities and directionality of filamentous-actin at the T cell immunological synapse, live-cell super-resolution imaging is combined with total internal reflection fluorescence and quantified with spatio-temporal image correlation spectroscopy.

קליפת המוח אקטין זרימה בתאי T לכמת ידי מתאם תמונת ספקטרוסקופיה מרחב ובזמן של תאורה מיקרוסקופית נתונים מובנים

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell ...

cortical-actin-flow-t-cells-quantified-spatio-temporal-image

Research

JoVE Journal

Immunology and Infection

9.6K Views.  King's College London. To investigate flow velocities and directionality of filamentous-actin at the T cell immunological synapse, live-cell super-resolution imaging is combined with total internal reflection fluorescence and quantified with spatio-temporal image correlation spectroscopy. 

Video: Cortical Actin Flow in T Cells Quantified by Spatio-temporal Image Correlation Spectroscopy of Structured Illumination Microscopy Data

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

התמרה retroviral של T-cell קולטנים עכבר מתאי T

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

שינוי מערכת Transposon piggyBac של תאי T אנושיים ראשוניים

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick tissue samples and organs in health and disease. By controlling the scanning pattern in multi-photon microscopy and applying appropriate numerical algorithms, we developed a striped-illumination approach, which enabled us to achieve 3-fold better axial resolution and improved signal-to-noise ratio, i.e. contrast, in more than 100 &#181;m tissue depth within highly scattering tissue of lymphoid organs as compared to standard multi-photon microscopy. The acquisition speed as well as photobleaching and photodamage effects were similar to standard photo-multiplier-based technique, whereas the imaging depth was slightly lower due to the use of field detectors. By using the striped-illumination approach, we are able to observe the dynamics of immune complex deposits on secondary follicular dendritic cells &#8211; on the level of a few protein molecules in germinal centers.

High-resolution intravital imaging with enhanced contrast up to 120 &#181;m depth in lymph nodes of adult mice is achieved by spatially modulating the excitation pattern of a multi-focal two-photon microscope. In 100 &#181;m depth we measured resolutions of 487 nm (lateral) and 551 nm (axial), thus circumventing scattering and diffraction limits.

מיקרוסקופית Intravital הפסים תאורה נפתר מאוד של מרכזי ז&#39;רמינל

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

אינדוקציה של Murine דלקת מעיים על ידי העברת המאמצת של מפעיל CD4<sup&gt; +</sup&gt; CD45RB<sup&gt; גבוה</sup&gt; תאי T לעכברי immunodeficient

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

T Cells Capture Bacteria by Transinfection from Dendritic Cells

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic cells (DCs) by a process called transinfection. Here, we describe the analysis of the transinfection process, which occurs during the course of antigen presentation. This process was unveiled by using CD4+ T cells from transgenic OTII mice, which bear a T cell receptor (TCR) specific for a peptide of ovoalbumin (OVAp), which therefore can form stable immune complexes with infected dendritic cells loaded with this specific OVAp. The dynamics of green fluorescent protein (GFP)-expressing bacteria during DC-T cell transmission can be monitored by live-cell imaging and the quantification of bacterial transinfection can be performed by flow cytometry. In addition, transinfection can be quantified by a more sensitive method based in the use of gentamicin, a non-permeable aminoglycoside antibiotic killing extracellular bacteria but not intracellular ones. This classical method has been used previously in microbiology to study the efficiency of bacterial infections. We hereby explain the protocol of the complete process, from the isolation of the primary cells to the quantification of transinfection.

Here a protocol is presented to measure bacterial capture by CD4+ T cells which occurs during antigen presentation via transinfection from pre-infected dendritic cells (DC). We show how to perform the necessary steps: isolation of primary cells, infection of DC, DC/T cell conjugate formation, and measurement of bacterial T cell transfection.

תאי T ללכוד חיידקים על ידי Transinfection מתאים דנדריטים

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic ...

The Use of Flow Cytometry to Assess the State of Chromatin in T Cells

During a proper immune response, quiescent T cells become activated upon antigen presentation to their antigen-specific T cell receptor. This leads to clonal proliferation of only those T cells that bear a receptor that recognizes the antigen. Chromatin decondensation is a hallmark of T cell activation and is required for T cells to acquire the ability to proliferate after antigen engagement. This change in chromatin condensation can be detected using antibodies raised against histone proteins. These antibodies cannot bind to their epitopes in na&#239;ve T cells as well as they can in activated T cells. We describe how to simultaneously stain T cell-specific surface markers, track viability with a fixable dead cell stain, and measure chromatin status via intracellular staining of Histone H3 proteins. Stained cells are analyzed by flow cytometry and chromatin condensation status is measured as the mean fluorescence intensity (MFI) of the Histone H3 stain. Chromatin decondensation during T cell activation is demonstrated as an increase in the MFI

Flow cytometry can be utilized to assess the state of chromatin within T cells. This protocol allows scientists to interpret evidence of chromatin decondensation during T cell activation demonstrated by an increase in the mean florescence intensity (MFI) of fluorescent Histone H3 antibodies

השימוש בcytometry הזרימה מנת להעריך את המצב של הכרומטין בתאי T

During a proper immune response, quiescent T cells become activated upon antigen presentation to their antigen-specific T cell receptor. This leads to ...

Preparation of CD4+ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy

The methods described herein for activation of na&#239;ve CD4+ T cells in suspension and their adherence in coverslips for confocal microscopy analysis allow the spatial localization and visualization of gangliosides involved in CD4+ T cell activation, that complement expression profiling experiments such as flow cytometry, western blotting or real-time PCR. The quantification of ganglioside expression through flow cytometry and their cellular localization through microscopy can be obtained by the use of anti-ganglioside antibodies with high affinity and specificity. Nonetheless, an adequate handling of cells in suspension involves the treatment of culture plates to promote the necessary adherence required for fluorescence or confocal microscopy acquisition. In this work, we describe a protocol for determining GD3 and GD2 ganglioside expression and colocalization with the TCR during na&#239;ve CD4+ T cell activation. Also, real-time PCR experiments using &#60;40,000 cells are described for the determination of the GD3 and GM2/GD2 synthase genes, demonstrating that gene analysis experiments can be performed with a low number of cells and without the need of additional low input RNA kits.

Preparation of CD4+ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy

We describe a standard antibody staining protocol for use in microscopy to determine the membrane expression and localization of gangliosides in resting and activated human na&#239;ve CD4+ T cells. Also described are real-time PCR experiments using &#60;40,000 cells that do not require additional low input RNA kits.

הכנת CD4<sup&gt; +</sup&gt; T תאים עבור ניתוח של ביטוי GD3 ו GD2 ganglioside ממברנה ידי מיקרוסקופית

The methods described herein for activation of na&#239;ve CD4+ T cells in suspension and their adherence in coverslips for confocal microscopy analysis ...

Molecular Diffusion in Plasma Membranes of Primary Lymphocytes Measured by Fluorescence Correlation Spectroscopy

Fluorescence correlation spectroscopy (FCS) is a powerful technique for studying the diffusion of molecules within biological membranes with high spatial and temporal resolution. FCS can quantify the molecular concentration and diffusion coefficient of fluorescently labeled molecules in the cell membrane. This technique has the ability to explore the molecular diffusion characteristics of molecules in the plasma membrane of immune cells in steady state (i.e., without processes affecting the result during the actual measurement time). FCS is suitable for studying the diffusion of proteins that are expressed at levels typical for most endogenous proteins. Here, a straightforward and robust method to determine the diffusion rate of cell membrane proteins on primary lymphocytes is demonstrated. An effective way to perform measurements on antibody-stained live cells and commonly occurring observations after acquisition are described. The recent advancements in the development of photo-stable fluorescent dyes can be utilized by conjugating the antibodies of interest to appropriate dyes that do not bleach extensively during the measurements. Additionally, this allows for the detection of slowly diffusing entities, which is a common feature of proteins expressed in cell membranes. The analysis procedure to extract molecular concentration and diffusion parameters from the generated autocorrelation curves is highlighted. In summary, a basic protocol for FCS measurements is provided; it can be followed by immunologists with an understanding of confocal microscopy but with no other previous experience of techniques for measuring dynamic parameters, such as molecular diffusion rates.

A method to measure protein diffusion in membranes of primary immune cells using fluorescence correlation spectroscopy (FCS) is described. In this paper, the use of antibodies for fluorescent labeling is illustrated.

דיפוזיה מולקולרית פלזמה ממברנות יסודי לימפוציטים נמדד באמצעות מתאם ספקטרוסקופיה פלואורסצנטי

Fluorescence correlation spectroscopy (FCS) is a powerful technique for studying the diffusion of molecules within biological membranes with high spatial ...

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore essential for understanding pathogenicity of different pathogens including HIV. Gamma delta (GD) T cells are the prototype of &#39;unconventional&#39; T cells and represent a relatively small subset of T cells defined by their expression of heterodimeric T-cell receptors (TCRs) composed of gamma and delta chains. This sets them apart from the classical and much better known CD4+ helper T cells and CD8+ cytotoxic T cells that are defined by alpha-beta TCRs. GD T cells often show tissue-specific localization and are enriched in epithelium. GD T cells orchestrate immune responses in inflammation, tumor surveillance, infectious disease, and autoimmunity.
Here, we present a method to reproducibly isolate and analyze human endocervical intraepithelial GD T lymphocytes. We have used endocervical cytobrush samples from women participating in the Women&#39;s Interagency HIV Infection Study (WIHS). Knowledge about GD T cells interactions during conditions in which there is an insult to the vaginal mucosal could be applied to any clinical study in which mucosal vulnerability is addressed, including the development of vaginal microbicides.In addition, knowledge about mucosal GD T cell responses has potential for application of GD T cell-based immune therapy in treating infectious diseases.

We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

ניתוח בידוד תזרים Cytometric של האדם תאים האנדוצרוויקאלית גמא דלתא T

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore ...

Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human central nervous system (CNS) autoimmune demyelinating disease, creation of an AQP4-targeted model with both clinical and histologic manifestations of CNS autoimmunity has proven challenging. Immunization of wild-type (WT) mice with AQP4 peptides elicited T cell proliferation, although those T cells could not transfer disease to na&#239;ve recipient mice. Recently, two novel AQP4 T cell epitopes, peptide (p) 135-153 and p201-220, were identified when studying immune responses to AQP4 in AQP4-deficient (AQP4-/-) mice, suggesting T cell reactivity to these epitopes is normally controlled by thymic negative selection. AQP4-/- Th17 polarized T cells primed to either p135-153 or p201-220 induced paralysis in recipient WT mice, that was associated with predominantly leptomeningeal inflammation of the spinal cord and optic nerves. Inflammation surrounding optic nerves and involvement of the inner retinal layers (IRL) were manifested by changes in serial optical coherence tomography (OCT). Here, we illustrate the approaches used to create this new in vivo model of AQP4-targeted CNS autoimmunity (ATCA), which can now be employed to study mechanisms that permit development of pathogenic AQP4-specific T cells and how they may cooperate with B cells in NMO pathogenesis.

Here, we present a protocol to induce paralysis and opticospinal inflammation by transfer of aquaporin-4 (AQP4)-specific T cells from AQP4-/- mice into WT mice. In addition, we demonstrate how to use serial optical coherence tomography to monitor visual system dysfunction.

אינדוקציה של שיתוק ופציעה מערכת הראייה אצל עכברים מאת T תאים ספציפיים עבור אופטיקה Neuromyelitis Autoantigen Aquaporin-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human ...