מחקר זה נתמך על ידי המחלקה למחקר המיפוי של המכון הלאומי לאלרגיה ומחלות זיהומיות, המכונים הלאומיים לבריאות. אנו מודים יוהנס החופה ומארק דיוויס לתת לנו scFv של H57 שימוש במחקרים אלה. RV רוצה להודות קיאר נוימן לדיון מועיל במהלך הפיתוח של טכניקה זו.

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כל בעלי החיים המשמשים בניסויים אלה נשמרו בסביבה הפתוגן ללא ספציפי, ואת הניסויים אושרו על ידי המכון הלאומי לבריאות בעלי חיים טיפול ועדת שימוש.

המתואר כאן מערכת ללמוד איתות אנטיגן ספציפי העיקריים טי תאים העכבר באמצעות TIRFM ותמך זכוכית השומנים bilayers כמו נגמ&quot;שים מלאכותיים. הטכניקה מתבססת על בהצלחה לבטא חלבונים GFP מתוייגים בתאים אלה. Transfecting ותאי T היא תמיד משימה מאתגרת. בדרך כלל, electroporation או הגן משלוח באמצעות רטרווירוסים lentiviruses או משמשים. אחת הטכניקות היא לא עדיפה על פני האחר, גם יש מגבלות ויתרונות שלהם. אנו מוצאים electroporation כי יש את היתרונות הבאים: 1) זה לא נדרש כי התאים להיות פעיל חלוקת כנדרש על רטרווירוסים אך לא lentiviruses, ו 2) רמת הביטוי יכול להיות נשלט על ידי שינוי זמן התאים מודגרת לפני הדמיה. החיסרון הגדול ביותר הוא שאנחנו מתקשים מאוד לבטא חלבונים כי הם גדולים בגודל של תאים ראשוניים. הצגנו שיטה נותן לנו כדאיות התא טוב מאוד אחרי electroporation. כתוצאה מכך הוא applicabלה להשתמש בו כדי להשיג בתיווך siRNA להשתקת גנים. כמו כן, אנו מתארים כאן מותאם אישית בו זמנית בערוץ 2 רכישת TIRF מיקרוסקופ, כי הוא תיקן chromatically ואינו דורש יישור נפרד עבור אורכי גל שונים. יכולות אלה, אך הם זמינים מסחרית. המערכת שלנו מבוססת על עקרונות של מיקרוסקופיה TIRF שפורסם על ידי מומחים בתחום, והוא זול יותר מאשר חלופות מסחריות. אחת הביקורת האפשרית של העיצוב שלנו הוא שאנחנו משתמשים באותה זווית של שכיחות עבור אורכי גל עירור שונים. הדבר יביא עומק החדירה TIRF שונה עבור אורכי גל עירור שונים. אנו סבורים כי תופעות אלה הן קטנות בגלל גל חולף הוא שדה מתפורר אקספוננציאלית ואת עומק השדה TIRF היא פונקציה ליניארית של אורך הגל. Fluorophores קרובים לפני השטח זכוכית יחוו הבדל בעוצמות לייזר בין שני אורכי גל. עבור fluorophores ליד PEעומק netration, ההבדל יהיה בעוצמה 1.3 לקפל את שני אורכי גל 488 ו 640 ננומטר 1.15 לקפל עבור שני אורכי גל 488 ו 561 ננומטר. אותה מערכת יכולה לשמש בשילוב עם טכניקות ברזולוציה סופר העושים שימוש בתאורה TIRF. תיארנו גם מערכת 2 המצלמה בנוי מותאם אישית. כמו מערכת TIRF, מנגנונים דומים זמינים גם מבחינה מסחרית. המערכת שלנו מציעה גמישות לשנות את המסננים ואת dichroics, המאפשר את השימוש בו עם שילובים שונים של אורכי גל. החיסרון של המערכת שלנו הוא מידת סיבוב אחד צריך ליישר את שתי התמונות. בעיה זו ניתן לפתור באמצעות עמדות מצלמה כי הם מונעים piezo ולהציע מעלות הסיבוב של יישור. יש לנו גם ליישם בהצלחה מערכת 2 המצלמה על מיקרוסקופ זה, שבו המצלמות הם שונים ויש להם גדלים שונים פיקסל. מערכת כזו תהיה יעילה אם היה צורך ברגישות בערוץ 1 ו dy גדולמגוון namic אחר, הן אלו לעתים נדירות זמין המצלמה זהה. השתמשנו במצלמה HQ-2 Photometrics ב binning 2x2 נותן לנו גודל פיקסל אפקטיבי של 12.9 מיקרומטר עם המצלמה Photometrics קוואנט-EM בעלת גודל פיקסל של 16 מיקרומטר. 180 מ&quot;מ אורך מוקד העדשה הצינור שימש קוואנט-EM ו 145 מ&quot;מ צינור מוקד העדשה אורך שימש המצלמה HQ-2. HQ-2 נשלט באמצעות Metamorph תוכנה קוואנט-EM נשלט באמצעות תוכנת מנהל מיקרו במחשב נפרד במצב טריגר חיצוני. טריגר חיצוני נמסר המצלמה קוואנט-EM באמצעות הפלט הדיגיטלי של לוח מחשוב DAC מדידה, אשר יושמה כמו תריס נוסף על קביעת תצורה של הגדרות תאורה Metamorph. היחס בין אורכי מוקד של עדשות צינור תואם את היחס בין הגדלים פיקסל אפקטיביים. יישור נציג מוצג באיור 5.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם מגיב </td><td> חברה </td><td> מספר קטלוגי </td><td> תגובות </td></tr><tr><td> Amaxa Nucleofector ערכת עכבר לתאי ה-T </td><td> Lonza בע&quot;מ </td><td> VPA-1006 </td><td></td></tr><tr><td> PureLink HiPure פלסמיד Midiprep Kit </td><td> החיים טכנולוגיות </td><td> K2100-05 </td><td> עבור Preps רעלן פנימי ללא ה-DNA </td></tr><tr><td> Amaxa התקן Nucleofector </td><td> Lonza בע&quot;מ </td><td> AAD-1001 </td><td></td></tr><tr><td> רקומביננטי Murine IL-2 </td><td> Peprotech בע&quot;מ </td><td> 212-12 </td><td></td></tr><tr><td> Fluoresbrite Multifluorescent microspheres 0.20μm </td><td> Polysciences, Inc </td><td> 24050-5 </td><td></td></tr><tr><td> Lab-Tek 8-II גם מחולק לתאים coverglass </td><td> Thermo Scientific, Nunc </td><td> 155409 </td><td></td></tr></tbody></table>

a tirf microscopy technique for real-time, simultaneous imaging of the tcr and its associated signaling proteins

איתות הוא יזם באמצעות קולטן תא T (TCR) כאשר היא עוסקת באמצעות פפטיד אנטיגני שברי מחויב קומפלקס Histocompatibility רס&quot;ן (pMHC) חלבונים לידי ביטוי על פני השטח של תאים מציגי אנטיגן (נגמ&quot;שים). הקומפלקס TCR מורכב heterodimer TCRαβ מחייב ליגנד המשייך לא קוולנטית עם CD3 הדימרים (את εδ ו εγ heterodimers ו ζζ homodimer) 1. על האירוסין של הקולטן, שרשראות ζ CD3 הם פוספורילציה ע&quot;י קינאז Src המשפחה, Lck. זה מוביל לגיוס של קינאז המשפחה Syk, Zap70, אשר פוספורילציה אז מופעל על ידי Lck. לאחר מכן, Zap70 phosphorylates חלבונים מתאם LAT ו SLP76, שיזם את הקמת מתחם איתות הפרוקסימלי המכיל מספר רב של מולקולות איתות שונות 2. היווצרות של זה בסופו של דבר התוצאות מורכבות בסידן ראס תלוי traגורם nscription הפעלה חניכה הסוגר של הסדרה מורכבת הביטוי תוכניות גנים שיוצרים בידול תא T 2. אותות (TCR לבין המדינה כתוצאה של בידול) הם מווסתת על ידי גורמים רבים אחרים, כולל עוצמה אנטיגן ו crosstalk עם co-stimulatory/co-inhibitory, chemokine, ואת קולטני ציטוקינים 3-4. לימוד הארגון במרחב ובזמן של המתחם איתות הפרוקסימלי בתנאי גירוי שונים היא, אם כן, המפתח להבנת מסלול איתות TCR כמו גם רגולציה על ידי במעברי האותות אחרים. מערכת אחת מודל שימושי מאוד ללמוד איתות ביוזמת TCR על הממברנה בתאי ה-T הוא הנתמכות זכוכית השומנים bilayers, כפי שתואר קודם לכן 5-6. הם יכולים להיות מנוצל כדי להציג pMHC אנטיגני קומפלקסים, הדבקה, ושיתוף גירוי מולקולות בתאי T-משמש נגמ&quot;שים מלאכותיים. על ידי הדמיה של תאים T אינטראקציה עםbilayer השומנים באמצעות הקרינה הכוללת פנימית השתקפות מיקרוסקופ (TIRFM), נוכל להגביל את עירור עד למרחק של 100 מייל ימי של שטח בין זכוכית לבין שטח פני התא 7-8. זה מאפשר לנו תמונה בעיקר את האירועים המתרחשים איתות קרום הפלזמה. כפי שאנו מעוניינים הדמיה לגיוס של איתות חלבונים מורכבים TCR, אנו מתארים מערכת של שתי המצלמה TIRF הדמיה שבה TCR, הנקרא Fab עם ניאון (אנטיגן שבר מחייב) שברי נוגדנים H57 (מטוהרים מן hybridoma H57-597 , ATCC, מספר ATCC: HB-218) שהוא ספציפי עבור TCRβ, ו איתות חלבונים, מתויג עם GFP, ניתן צילמו בו זמנית ובזמן אמת. אסטרטגיה זו הכרחית בשל האופי הדינמי ביותר של שני תאים T ו האירועים איתות שמתרחשים ב TCR. שיטת הדמיה זו אפשרה לחוקרים ligands תמונה יחידה 9-11, כמו גם גיוס של מולקולות איתות לקולטנים מופעלהיא מערכת מצוינת ללמוד ביוכימיה באתרו 12-16.

Watch this Scientific Journal Video about A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins at JoVE.com

A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins

המידור של חלבונים גם בתוך קרום פלזמה או אל מקומות תאיים הוא אחד מנגנון רגולטורי כי יכול מאוד להשפיע על התוצאות איתות, ולכן, כדי להבין איתות חשוב ללמוד את התנהגות במרחב ובזמן של חלבונים המעורבים. המתואר כאן מערכת מבוססת מיקרוסקופיה TIRF ללמוד התמרה האות בתאי T, אבל הוא החלים רחב.

טכניקה מיקרוסקופית TIRF הדמיה בזמן אמת סימולטני של TCR וחלבונים הקשורים אליו לשידור אותות

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex ...

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Universidad de Cartagena UDEC

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JoVE Journal

Immunology and Infection

23.8K Views.  National Institute of Allergy and Infectious Diseases, National Institutes of Health. המידור של חלבונים גם בתוך קרום פלזמה או אל מקומות תאיים הוא אחד מנגנון רגולטורי כי יכול מאוד להשפיע על התוצאות איתות, ולכן, כדי להבין איתות חשוב ללמוד את התנהגות במרחב ובזמן של חלבונים המעורבים. המתואר כאן מערכת מבוססת מיקרוסקופיה TIRF ללמוד התמרה האות בתאי T, אבל ה...

Video: A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &beta;-arrestin

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to intracellular responses. GPCRs control diverse biological functions such as chemotaxis, intracellular calcium release, gene regulation in a ligand dependent manner via heterotrimeric G-proteins1-2. Ligand binding induces a series of conformational changes leading to activation of heterotrimeric G-proteins that modulate levels of second messengers such as cyclic adenosine monophosphate (cAMP), inositol triphosphate (IP3) and diacyl glycerol (DG). Concomitant with activation of the receptor ligand binding also initiates a series of events to attenuate the receptor signaling via desensitization, sequestration and/or internalization. The desensitization process of GPCRs occurs via receptor phosphorylation by G-protein receptor kinases (GRKs) and subsequent binding of &beta;-arrestins3. &beta;-arrestins are cytosolic proteins and translocate to membrane upon GPCR activation, binding to phosphorylated receptors (most cases) there by facilitating receptor internalization 4-6.
Leukotriene B4 (LTB4) is a pro-inflammatory lipid molecule derived from arachidonic acid pathway and mediates its actions via GPCRs, LTB4 receptor 1 (BLT1; a high affinity receptor) and LTB4 receptor 2 (BLT2; a low affinity receptor)7-9. The LTB4-BLT1 pathway has been shown to be critical in several inflammatory diseases including, asthma, arthritis and atherosclerosis10-17. The current paper describes the methodologies developed to monitor LTB4-induced leukocyte migration and the interactions of BLT1 with &beta;-arrestin and , receptor translocation in live cells using microscopy imaging techniques18-19.
Bone marrow derived dendritic cells from C57BL/6 mice were isolated and cultured as previously described 20-21. These cells were tested in live cell imaging methods to demonstrate LTB4 induced cell migration. The human BLT1 was tagged with red fluorescent protein (BLT1-RFP) at C-terminus and &beta;-arrestin1 tagged with green fluorescent protein (&beta;-arr-GFP) and transfected the both plasmids into Rat Basophilic Leukomia (RBL-2H3) cell lines18-19. The kinetics of interaction between these proteins and localization were monitored using live cell video microscopy. The methodologies in the current paper describe the use of microscopic techniques to investigate the functional responses of G-protein coupled receptors in live cells. The current paper also describes the use of Metamorph software to quantify the fluorescence intensities to determine the kinetics of receptor and cytosolic protein interactions.

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &#946;-arrestin

This paper describes the methodology to determine the chemotactic response of leukocytes to specific ligands and identify interactions between the cell surface receptors and cytosolic proteins using live cell imaging techniques.

בזמן אמת הדמיה של leukotriene B 4 Cell Migration מתווכת ו BLT1 אינטראקציות עם β-arrestin

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to ...

Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse

The lymphatic vascular system is an important component of the circulatory system that maintains fluid homeostasis, provides immune surveillance, and mediates fat absorption in the gut. Yet despite its critical function, there is comparatively little understanding of how the lymphatic system adapts to serve these functions in health and disease1. Recently, we have demonstrated the ability to dynamically image lymphatic architecture and lymph "pumping" action in normal human subjects as well as in persons suffering lymphatic dysfunction using trace administration of a near-infrared fluorescent (NIRF) dye and a custom, Gen III-intensified imaging system2-4. NIRF imaging showed dramatic changes in lymphatic architecture and function with human disease. It remains unclear how these changes occur and new animal models are being developed to elucidate their genetic and molecular basis. In this protocol, we present NIRF lymphatic, small animal imaging5,6 using indocyanine green (ICG), a dye that has been used for 50 years in humans7, and a NIRF dye-labeled cyclic albumin binding domain (cABD-IRDye800) peptide that preferentially binds mouse and human albumin8. Approximately 5.5 times brighter than ICG, cABD-IRDye800 has a similar lymphatic clearance profile and can be injected in smaller doses than ICG to achieve sufficient NIRF signals for imaging8. Because both cABD-IRDye800 and ICG bind to albumin in the interstitial space8, they both may depict active protein transport into and within the lymphatics. Intradermal (ID) injections (5-50 &mu;l) of ICG (645 &mu;M) or cABD-IRDye800 (200 &mu;M) in saline are administered to the dorsal aspect of each hind paw and/or the left and right side of the base of the tail of an isoflurane-anesthetized mouse. The resulting dye concentration in the animal is 83-1,250 &mu;g/kg for ICG or 113-1,700 &mu;g/kg for cABD-IRDye800. Immediately following injections, functional lymphatic imaging is conducted for up to 1 hr using a customized, small animal NIRF imaging system. Whole animal spatial resolution can depict fluorescent lymphatic vessels of 100 microns or less, and images of structures up to 3 cm in depth can be acquired9. Images are acquired using V++ software and analyzed using ImageJ or MATLAB software. During analysis, consecutive regions of interest (ROIs) encompassing the entire vessel diameter are drawn along a given lymph vessel. The dimensions for each ROI are kept constant for a given vessel and NIRF intensity is measured for each ROI to quantitatively assess "packets" of lymph moving through vessels.

Recently developed imaging techniques using near-infrared fluorescence (NIRF) may help elucidate the role the lymphatic system plays in cancer metastasis, immune response, wound repair, and other lymphatic-associated diseases.

דימות אופטי לא פולשנית של כלי דם הלימפה של עכבר

The lymphatic  vascular system is an important component of the circulatory system that  maintains fluid homeostasis, provides immune surveillance, and ...

Real-time Live Imaging of T-cell Signaling Complex Formation

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating the activation and responses of multiple immune cells 3,4. T-cell activation is dependent on the recognition of specific antigens displayed by antigen presenting cells (APCs). The T-cell antigen receptor (TCR) is specific to each T-cell clone and determines antigen specificity 5. The binding of the TCR to the antigen induces the phosphorylation of components of the TCR complex. In order to promote T-cell activation, this signal must be transduced from the membrane to the cytoplasm and the nucleus, initiating various crucial responses such as recruitment of signaling proteins to the TCR;APC site (the immune synapse), their molecular activation, cytoskeletal rearrangement, elevation of intracellular calcium concentration, and changes in gene expression 6,7. The correct initiation and termination of activating signals is crucial for appropriate T-cell responses. The activity of signaling proteins is dependent on the formation and termination of protein-protein interactions, post translational modifications such as protein phosphorylation, formation of protein complexes, protein ubiquitylation and the recruitment of proteins to various cellular sites 8. Understanding the inner workings of the T-cell activation process is crucial for both immunological research and clinical applications.
Various assays have been developed in order to investigate protein-protein interactions; however, biochemical assays, such as the widely used co-immunoprecipitation method, do not allow protein location to be discerned, thus precluding the observation of valuable insights into the dynamics of cellular mechanisms. Additionally, these bulk assays usually combine proteins from many different cells that might be at different stages of the investigated cellular process. This can have a detrimental effect on temporal resolution. The use of real-time imaging of live cells allows both the spatial tracking of proteins and the ability to temporally distinguish between signaling events, thus shedding light on the dynamics of the process 9,10. We present a method of real-time imaging of signaling-complex formation during T-cell activation. Primary T-cells or T-cell lines, such as Jurkat, are transfected with plasmids encoding for proteins of interest fused to monomeric fluorescent proteins, preventing non-physiological oligomerization 11. Live T cells are dropped over a coverslip pre-coated with T-cell activating antibody 8,9, which binds to the CD3/TCR complex, inducing T-cell activation while overcoming the need for specific activating antigens. Activated cells are constantly imaged with the use of confocal microscopy. Imaging data are analyzed to yield quantitative results, such as the colocalization coefficient of the signaling proteins.

We describe a live-cell imaging method that provides insight into protein dynamics during the T-cell activation process. We demonstrate the combined usage of the T-cell spreading assay, confocal microscopy and imaging analysis to yield quantitative results to follow signaling complex formation throughout T-cell activation.

הדמית חיה בזמן אמת של T-תא איתות גיבוש קומפלקס

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating ...

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 ...

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

הדמיה של Dynamics התאים מיאלואידית בזמן אמת<em&gt; Apc<sup&gt; מינימום / +</sup</em&gt; מעיים גידולים על ידי דיסק מסתובב Confocal מיקרוסקופית

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

Immunofluorescence to Monitor the Cellular Uptake of Human Lactoferrin and its Associated Antiviral Activity Against the Hepatitis C Virus

Immunofluorescence is a laboratory technique commonly used to study many aspects of biology. It is typically used to visualize the distribution and/or localization of a target molecule in cells and tissues. Immunofluorescence relies on the specificity of fluorescent-labelled antibodies against their corresponding antigens within a cell. Both direct and indirect immunofluorescence approaches can be used which rely on the use of antibodies linked with a fluorochrome. Direct immunofluorescence is less frequently used because it provides lower signal, involves higher cost and less flexibility. In contrast, indirect immunofluorescence is more commonly used because of its high sensitivity and provides an amplified signal since more than one secondary antibody can attach to each primary antibody. In this manuscript, both epifluorescence microscopy and confocal microscopy were used to monitor the internalization of human lactoferrin, an important component of the immune system, into hepatic cells. Moreover, we monitored the inhibitory potential of hLF on the intracellular replication of the Hepatitis C virus using immunofluorescence. Both the advantages and disadvantages associated with these approaches are discussed.

The human lactoferrin (hLF) is a component of the immune system. In this study, immunofluorescence assays are used to demonstrate both the hepatocellular uptake of hLF and a qualitative reduction in the hepatitis C virus replication upon treatment with hLF.

Immunofluorescence לפקח על הספיגה הסלולארית של לקטופרין האדם והפעילות הקשורה אנטי נגד וירוס הצהבת מסוג C

Immunofluorescence is a laboratory technique commonly used to study many aspects of biology. It is typically used to visualize the distribution and/or ...

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target cells. Inside the host cell the effector proteins manipulate cellular functions to the benefit of the bacteria. To better understand the control of type III secretion during host cell interaction, sensitive and accurate assays to measure translocation are required. We here describe the application of an assay based on the fusion of a Yersinia enterocolitica effector protein fragment (Yersinia outer protein; YopE) with TEM-1 beta-lactamase for quantitative analysis of translocation. The assay relies on cleavage of a cell permeant FRET dye (CCF4/AM) by translocated beta-lactamase fusion. After cleavage of the cephalosporin core of CCF4 by the beta-lactamase, FRET from coumarin to fluorescein is disrupted and excitation of the coumarin moiety leads to blue fluorescence emission. Different applications of this method have been described in the literature highlighting its versatility. The method allows for analysis of translocation in vitro and also in in vivo, e.g., in a mouse model. Detection of the fluorescence signals can be performed using plate readers, FACS analysis or fluorescence microscopy. In the setup described here, in vitro translocation of effector fusions into HeLa cells by different Yersinia mutants is monitored by laser scanning microscopy. Recording intracellular conversion of the FRET reporter by the beta-lactamase effector fusion in real-time provides robust quantitative results. We here show exemplary data, demonstrating increased translocation by a Y. enterocolitica YopE mutant compared to the wild type strain.

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Effector translocation into host cells via a type III secretion system is a common virulence strategy among gram-negative bacteria. A beta-lactamase effector fusion based assay for quantitative analysis of translocation was applied. In Yersinia infected cells, conversion of a FRET reporter by the beta-lactamase is monitored using laser scanning microscopy.

ניתוח של<em&gt; Enterocolitica Yersinia</em&gt; מפעיל טרנסלוקציה לתאי מארח שימוש בטא לקטמאז מפעיל Fusions

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target ...

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in vivo is crucial for understanding the molecular basis of leukocyte transendothelial migration and interaction with vascular endothelium. One powerful approach involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.
Here we present a protocol for imaging monocytes and neutrophils in the CX3CR1gfp/wt mouse i.v. injected with orange dye-labeled neutrophils with an inverted confocal microscope. Time-lapse movies gathered from 30 min&#160;to several hours of imaging allow the analysis of leukocyte behavior in mesenteric veins under both steady state and inflammatory conditions. We also describe the steps to locally induce blood vessel inflammation with TLR2/TLR1 agonist Pam3SK4 and monitor the subsequent recruitment of neutrophils and monocytes.
The presented technique can also be used to monitor other populations of leukocytes and investigate molecules implicated in leukocyte recruitment or trafficking using other stimuli or transgenic mice.

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

הדמיה נויטרופילים ומונוציטים בmesenteric ורידים על ידי מיקרוסקופית Intravital על עכברים מורדמים בזמן אמת

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and cellular proteins by mass spectrometry. Although proteins that interact with viral genomes play major roles in determining the outcome of infection, a comprehensive analysis of viral genome associated proteins was not previously feasible. Here we demonstrate a method that enables the direct purification of HSV-1 genomes from infected cells. Replicating viral DNA is selectively labeled with modified nucleotides that contain an alkyne functional group. Labeled DNA is then specifically and irreversibly tagged via the covalent attachment of biotin azide via a copper(I)-catalyzed azide-alkyne cycloaddition or click reaction. Biotin-tagged DNA is purified on streptavidin-coated beads and associated proteins are eluted and identified by mass spectrometry. This method enables the selective targeting and isolation of HSV-1 replication forks or whole genomes from complex biological environments. Furthermore, adaptation of this approach will allow for the investigation of various aspects of herpesviral infection, as well as the examination of the genomes of other DNA viruses.

The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins.

טיהור של ה-DNA הנגיפי לצורך זיהוי חלבונים הקשורים ויראלי וסלולריות

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and ...

Real-time Imaging and Quantification of Fungal Biofilm Development Using a Two-Phase Recirculating Flow System

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. While models for the growth under flow have been developed, many of these systems are expensive, or do not allow imaging while the cells are under flow. We have developed a novel apparatus that allows us to image the growth and development of Candida albicans cells under flow and in real-time. Here, we detail the protocol for the assembly and use of this flow apparatus, as well as the quantification of data that are generated. We are able to quantify the rates that the cells attach to and detach from the slide, as well as to determine a measure of the biomass on the slide over time. This system is both economical and versatile, working with many types of light microscopes, including inexpensive benchtop microscopes, and is capable of extended imaging times compared to other flow systems. Overall, this is a low-throughput system that can provide highly detailed real-time information on the biofilm growth of fungal species under flow.

We describe the assembly, operation, and cleaning of a flow apparatus designed to image fungal biofilm formation in real time while under flow. We also provide and discuss quantitative algorithms to be used on the acquired images.

הדמיה בזמן אמת, כימות של פיתוח Biofilm פטרייתי באמצעות מערכת זרימת Recirculating דו-שלבית

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. ...

טכניקה מיקרוסקופית TIRF הדמיה בזמן אמת סימולטני של TCR וחלבונים הקשורים אליו לשידור אותות

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בזמן אמת הדמיה של leukotriene B₄ Cell Migration מתווכת ו BLT1 אינטראקציות עם β-arrestin