אנו מודים ג&#39;Sedgewick לסיוע בהקמת מיקרוסקופ confocal ללכוד תמונות של גרגרי כסף; נתמך בחלקו על ידי מענקי מחקר NIH AI48484 (ATH), AI20048 (ע&quot;ר), ו AI066314 (CJM); המרכז הלאומי לחקר משאבי מענק. RR00169 (CNPRC, מאוניברסיטת קליפורניה, דייוויס).

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	<li>Altman, J. D., Moss, P. A. H., Goulder, P. J. R., Barouch, D. H., McHeyzer-Williams, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+analysis+of+antigen-specific+T+lymphocytes.">Phenotypic analysis of antigen-specific T lymphocytes.</a> Science. 274, 94-96 (1996).</li><li>Skinner, P. J., Daniels, M. A., Schmidt, C. S., Jameson, S. C., Haase, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutting+Edge:+In+situ+tetramer+staining+of+antigen-specific+T+cells+in+tissues.">Cutting Edge: In situ tetramer staining of antigen-specific T cells in tissues.</a> Journal of Immunology. 165, 617-61 (2000).</li><li>Qingsheng, L. i., Skinner, P. J., Ha, S. -. J., Duan, L., Mattila, T., Hage, A., White, C., Barber, D. L., O'mara, L., Southern, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+antigen-specific+and+infected+cells+in+situ+predicts+outcome+in+early+viral+infection.">Visualizing antigen-specific and infected cells in situ predicts outcome in early viral infection.</a> Science. 323, 1726-1729 (2008).</li><li>Haase, T., Haase, K., Zupancic, M., Sedgewick, G., Faust, R. A., Melroe, H., Cavert, W., Gebhard, K., Staskus, K., Zhang, Z. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perelson+Quantitative+Image+Analysis+of+HIV-1+Infection+in+Lymphoid+Tissue.">Perelson Quantitative Image Analysis of HIV-1 Infection in Lymphoid Tissue.</a> Science. 274, 985-989 (1996).</li><li>Li, Q., Duan, L., Estes, J. D., Ma, Z. M., Rourke, T., Wang, Y., Reilly, C., Carlis, J., Miller, C. J., Haase, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peak+SIV+replication+in+resting+memory+CD4+T+cells+depletes+gut+lamina+propria+CD4+T+cells.">Peak SIV replication in resting memory CD4 T cells depletes gut lamina propria CD4 T cells.</a> Nature. 434, 1148-1152 (2005).</li><li>Li, Q. i. n. g. s. h. e. n. g., Estes, J. a. c. o. b. D., Schlievert, P. a. t. r. i. c. k. M., Duan, L. i. j. i. e., Brosnahan, A. m. a. n. d. a. J., Southern, P. e. t. e. r. J., Reilly, C. a. v. a. n. S., Peterson, M. a. r. n. i. e. L., Schultz-Darken, N. a. n. c. y., Brunner, K. e. v. i. n. G., Nephew, K. a. r. l. a. R., Pambuccian, S. t. e. f. a. n., Lifson, J. e. f. f. r. e. y. D., &amp;amp, J. o. h. n. V. C. a. r. l. i. s., Haase, A. s. h. l. e. y. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycerol+monolaurate+prevents+mucosal+SIV+transmission.">Glycerol monolaurate prevents mucosal SIV transmission.</a> Nature. , (2009).</li></ol>

The authors have nothing to disclose.

מאז נגיף RNA משמש כסמן תאים הנגועים בנגיף בדוח זה, כל ריאגנטים לפני הכלאה צריכים להיות חופשיים RNAase. בהליך הכלאה באתרו המתואר כאן שונתה כדי לשמר את האות ניאון מ ב מכתים tetramer באתרה. מאז האות radioautographic מ הנגועים בנגיף תאים זוהה על ידי הכלאה ב באתרו הוא בעומק של בערך בקוטר-תאים, בסעיפים עבה המשמש ב מכתים tetramer באתרו להיות חתכה מחדש תוך 5-8 מיקרון בעובי חלקים. זו טכניקה חדשנית של שילוב של tetramers באתרו של הכלאה באתרו (ISTH) המתואר כאן מאפשר מיפוי להדמיה סימולטני, וניתוח הקשר המרחבי של וירוס ספציפי בתאי CD8 + T ו-נגוע בווירוס התאים היעד. שיטה זו יכולה להיות מיושמת על מנת להעריך CD8 + T תא החיסון מבוסס על נגיף פתוגן שאינו אחרים ואת האינטראקציות המארח. שיטת ניתוח התמונה שתוארה כאן יכול להיות גם מותאם המחקר יחסים מרחביים של כל שני תאים אוכלוסיות אינטראקציה באתרו, למשל, אנחנו לאחרונה השתמשו הליך מיפוי centroid בלימוד של העברת HIV-1 המינית במודל פרימטים לא אנושיים של חסר חיסוני הקופי זיהום וירוס של קופי רזוס 6

פתרונות ריאגנטים: <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> PBS-H פוספט שנאגרו הפרין המכיל תמיסת מלח (100ug/ml או 18.7U/ml) לעכב RNASes </li><li style=";text-align:right;direction:rtl"> תרבות RPMI רקמות מדיה המכילה הפרין (100ug/ml או 18.7U/ml) לעכב RNASes </li><li style=";text-align:right;direction:rtl"> 4% נמוך להמיס agarose ב PBS </li><li style=";text-align:right;direction:rtl"> PBS עם סרום 2% עז רגילה (או בסרום ממינים שבה נוגדנים מצומדות fluorophore נעשו). </li><li style=";text-align:right;direction:rtl"> PBS עם סרום 2% עז נורמלי טריטון 0.3% X-100 </li><li style=";text-align:right;direction:rtl"> בכיתה MHC אני tetramers מצומדות כדי FITC </li><li style=";text-align:right;direction:rtl"> Anti-FITC נוגדנים, למשל Biodesign ארנבת אנטי FITC </li><li style=";text-align:right;direction:rtl"> מצומדות Fluorophore נגד ארנב IgG כי כבר מאוד לחצות adsorbed למינים אחרים IgG לשימוש עם תיוג מרובות, למשל עזים נגד ארנב Cy3 </li><li style=";text-align:right;direction:rtl"> מצומדות Fluorophore אנטי עכבר IgG כי כבר מאוד לחצות adsorbed למינים אחרים IgG לשימוש עם תיוג מרובים </li><li style=";text-align:right;direction:rtl"> טרי PBS שנאגרו paraformaldehyde 4% </li><li style=";text-align:right;direction:rtl"> 0.01M דשן </li><li style=";text-align:right;direction:rtl"> גליצרול / הג&#39;לטין המכיל 4mg/ml n-propyl gallate </li></ul> ציוד מיוחד <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> Vibratome </li><li style=";text-align:right;direction:rtl"> אזמל נתוחים </li><li style=";text-align:right;direction:rtl"> סכיני גילוח </li><li style=";text-align:right;direction:rtl"> כירורגי מספריים </li><li style=";text-align:right;direction:rtl"> Loctite הגדר מהירה מיידית דבק (מוצר # 46551) </li><li style=";text-align:right;direction:rtl"> מצבטים </li><li style=";text-align:right;direction:rtl"> # 2 מברשות צבע שיער גמלים. אפשר לקשט עם סכין גילוח לעובי הרצוי </li><li style=";text-align:right;direction:rtl"> 24 גם שטוח רקמה תחתית תרבות צלחות למשל פלקון קטלוג # 353226 </li><li style=";text-align:right;direction:rtl"> לתאי רקמות </li><li style=";text-align:right;direction:rtl"> טין לסכל </li><li style=";text-align:right;direction:rtl"> תיקיית קרטון שקופיות למשל פישר קטלוג # 12-587-10 </li><li style=";text-align:right;direction:rtl"> Confocal מיקרוסקופ </li></ul> IST ריאגנטים לעיון מהיר PBS-H (פוספט בופר סליין עם הפרין) <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> 450 מ&quot;ל 1X PBS </li><li style=";text-align:right;direction:rtl"> 50 מ&quot;ל 1X PBS + הפרין 10X </li></ul> 1x PBS + הפרין 10X <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> 500 מ&quot;ל 1X PBS </li><li style=";text-align:right;direction:rtl"> 500 מ&quot;ג אבקת הפרין (נמצא על המדף כימי יבש אחסון) </li></ul> NGS PBS-H / 2% (עז נורמלי בסרום) <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> 49 מ&quot;ל PBS-H של פלקון צינור </li><li style=";text-align:right;direction:rtl"> 1 מ&quot;ל NGS (נמצאו 1 aliquots מ&quot;ל במקפיא -20) </li></ul> NGS PBS-H / 2% / 0.3% Triton X-100 <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> 49 מ&quot;ל PBS-H / טריטון 0.3% X-100 ב פלקון צינור </li><li style=";text-align:right;direction:rtl"> 1 מ&quot;ל NGS (נמצאו 1 aliquots מ&quot;ל במקפיא -20) </li></ul> PBS-H / 0.3% Triton X-100 <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> 500 מ&quot;ל PBS-H </li><li style=";text-align:right;direction:rtl"> 1.5 מ&quot;ל טריטון X-100 (זהו נוזל סמיך למצוא על מדפי אחסון יבש כימי) </li></ul> 4% LMP agarose <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> 2 גרם אבקת LMP agarose </li><li style=";text-align:right;direction:rtl"> 50 מ&quot;ל PBS </li><li style=";text-align:right;direction:rtl"> Shake עד מיקרוגל, מעורב עד להמסת האבקה. </li></ul> GG-NPG (גליצרול ג&#39;לטין עם n-propyl galate <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מניחים בקבוק של גליצרול ג&#39;לטין באמבט 50 מעלות מים כדי להמיס </li><li style=";text-align:right;direction:rtl"> הוסף 0.06 גר &#39;של propyl galate (נמצא על המדף טריס), לנער היטב </li><li style=";text-align:right;direction:rtl"> שמור באמבט מים, רועד מדי פעם עד שהיא נמסה. </li><li style=";text-align:right;direction:rtl"> Aliquot לתוך צינורות 1 מ&quot;ל (צינורות חום) </li></ul> 4% paraformaldehyde <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> 4 גרם אבקת paraformaldehyde (נמצא על המדפים אחסון יבש כימי) </li><li style=";text-align:right;direction:rtl"> שים על 10 מ&quot;ל PBS-H בתוך גליל סיימה, להוסיף אבקת PF </li><li style=";text-align:right;direction:rtl"> מוסיפים מעט מים עד נפח מגיע לכ 70 מ&quot;ל </li><li style=";text-align:right;direction:rtl"> הוסף אחת של טפטפת NaOH. </li><li style=";text-align:right;direction:rtl"> מערבבים עד שהיא נמסה (בדרך כלל כ - 20-30 דקות) </li><li style=";text-align:right;direction:rtl"> הוסף HCl עד pH מגיע בין 7-8. </li><li style=";text-align:right;direction:rtl"> תביאו נפח 100 מ&quot;ל מים. </li></ul> דשן 0.01 מ &#39; <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> 0.3 גרם אוריאה (נמצא על מדפי אחסון יבש כימי) </li><li style=";text-align:right;direction:rtl"> 500 מ&quot;ל מים </li></ul>

a technique to simultaneously visualize virus-specific cd8+ t cells and virus-infected cells in situ

המספרים של הנגיף במקומות ספציפיים CD8 + T תאים ביחס מספרים ומיקומים של מטרות נגועים שלהם התא נחשב קריטי בקביעת התוצאות אשר נע בין פינוי מתמשך לזיהומים כרוניים. אנו מתארים כאן שיטה להערכת יחסים מרחביים כמותיים בין החיסון מפעיל (ה) וירוס ספציפי CD8 + T תאים מטרות נגוע (T) אשר משלב מכתים באתרו (IST) tetramer לזהות וירוסים ספציפיים CD8 + T תאים ו באתרו ההכלאה (שאני עורך) כדי לזהות נגיפי-RNA + תאים ברקמות שם את הקרב בין ההגנה החיסונית וזיהום מתרחש. השילוב של מכתים IST ו - איש, מקוצר ISTH, מאפשרת הדמיה ומיפוי של המיקומים של תאים מפעיל מטרות החיסונית, ונחישות קליל של E: יחסי טי. פרמטרים אלה בתורם לאחר מכן ניתן להשתמש כדי לקבוע את יחסי הגומלין בין קרבה מרחבית, ואת העיתוי ואת העוצמה של התגובה החיסונית כי לחזות תוצאות של זיהום מוקדם.

Watch this Scientific Journal Video about A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells In situ at JoVE.com

A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells In situ

טכניקה המשלבת ב מכתים tetramer באתרו של הכלאה באתרו (ISTH) מאפשר, הדמיית מיפוי וניתוח של הקרבה המרחבי של ספציפיים וירוס CD8 תאים + T ליעדים הנגועים בנגיף שלהם, וקביעת היחסים הכמותיים בין effectors מטרות החיסונית לתוצאות זיהום.

טכניקה במקביל דמיינו ספציפיות וירוס CD8 + T ו-תאים הנגועים בווירוס תאים באתרו</em

The numbers and locations of virus-specific CD8+ T cells relative to the numbers and locations of their infected cell targets is thought to be critical in ...

a-technique-to-simultaneously-visualize-virus-specific-cd8-t-cells

Research

JoVE Journal

Biology

A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells In situ

12.0K Views.  University of Minnesota. טכניקה המשלבת ב מכתים tetramer באתרו של הכלאה באתרו (ISTH) מאפשר, הדמיית מיפוי וניתוח של הקרבה המרחבי של ספציפיים וירוס CD8 תאים + T ליעדים הנגועים בנגיף שלהם, וקביעת היחסים הכמותיים בין effectors מטרות החיסונית לתוצאות זיהום.

Video: A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells In situ

Regulatory T cells: Therapeutic Potential for Treating Transplant Rejection and Type I Diabetes

רגולציה T תאים: הפוטנציאל הטיפולי לטיפול דחייה וסוג אני סוכרת

Homing of Hematopoietic Cells to the Bone Marrow

Homing is the phenomenon whereby transplanted hematopoietic cells are able to travel to and engraft or establish residence in the bone marrow. Various chemomkines and receptors are involved in the homing of hematopoietic stem cells. [1, 2]
This paper outlines the classic homing protocol used in hematopoietic stem cell studies. In general this involves isolating the cell population whose homing needs to be investigated, staining this population with a dye of interest and injecting these cells into the blood stream of a recipient animal. The recipient animal is then sacrificed at a pre-determined time after injection and the bone marrow evaluated for the percentage or absolute number of cells which are positive for the dye of interest. In one of the most common experimental schemes, the homing efficiency of hematopoietic cells from two genetically distinct animals (a wild type animal and the corresponding knock-out) is compared. This article describes the hematopoietic cell homing protocol in the framework of such as experiment.

This article describes a protocol used to study the homing of hematopoietic cells to their niches in the bone marrow.

ביות של תאים hematopoietic אל מח העצם

Homing is the phenomenon whereby transplanted hematopoietic cells are able to travel to and engraft or establish residence in the bone marrow.  Various ...

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, many proteins are present as a freely diffusing population in free exchange with a sub-population that is tightly associated with a particular subcellular domain or structure. In situ subcellular fractionation allows the visualization of protein compartmentalization and can also reveal protein sub-populations that localize to specific structures. For example, removal of soluble cytoplasmic proteins and loosely held nuclear proteins can reveal the stable association of some transcription factors with chromatin. Subsequent digestion of DNA can in some cases reveal association with the network of proteins and RNAs that is collectively termed the nuclear scaffold or nuclear matrix. 
Here we describe the steps required during the in situ fractionation of adherent and non-adherent mammalian cells on microscope coverslips. Protein visualization can be achieved using specific antibodies or fluorescent fusion proteins and fluorescence microscopy. Antibodies and/or fluorescent dyes that act as markers for specific compartments or structures allow protein localization to be mapped in detail. In situ fractionation can also be combined with western blotting to compare the amounts of protein present in each fraction. This simple biochemical approach can reveal associations that would otherwise remain undetected.

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

In situ subcellular fractionation of mammalian cells on microscope coverslips allows the visualisation of protein localisation.

ב ההיפוך חלוקה subcellular באתרו של בתאי יונקים חסיד שאינן חסיד

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, ...

Isolation of Valvular Endothelial Cells

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system. These thin, fibrous tissues are subjected to significant mechanical stresses as they open and close several billion times over a lifespan. The incredible endurance of these tissues is due to the resident valvular endothelial (VEC) and interstitial cells (VIC) that constantly repair and remodel in response to local mechanical and biological signals. Only recently have we begun to understand the unique behaviors of these cells, for which in vitro experimentation has played a key role. Particularly challenging is the isolation and culture of VEC. Special care must be used from the moment the tissue is removed from the host through final plating. Here we present protocols for direct isolation, side specific isolation, culture, and verification of pure populations of VEC. We use enzymatic digestion followed by a gentle swab scraping technique to dislodge only surface cells. These cells are then collected into a tube and centrifuged into a pellet. The pellet is then resuspended and plated into culture flasks pre-coated with collagen I matrix. VEC phenotype is confirmed by contact inhibited growth and the expression of endothelial specific markers such as PECAM1 (CD31), Von Willebrand Factor (vWF), and negative expression of alpha-smooth muscle actin (&alpha;-SMA). The functional characteristics of VEC are associated with high levels of acetylated LDL. Unlike vascular endothelial cells, VEC have the unique capacity to transform into mesenchyme, which normally occurs during embryonic valve formation1. This can also occur during significantly prolonged post confluent in vitro culture, so care should be made to passage at or near confluence. After VEC isolation, pure populations of VIC can then be easily acquired.

We provide a method for isolating and culturing pure populations of heart valve endothelial cells (VEC). VEC can be isolated from either side of the cusp or leaflet and immediately following, underlying interstitial cell (VIC) isolation is straightforward.

בידוד של תאים אנדותל valvular

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system.  These thin, fibrous tissues are ...

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

Assay Clonogenic: תאים חסיד

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated precursors. Researchers have employed various methods of lineage labeling, such as DiI labeling1 and pressure injection of traceable enzymes2 to ascertain cell fate at later stages of development in model systems. The first fate maps in zebrafish (Danio rerio) were assembled by iontophoretic injection of fluorescent dyes, such as rhodamine dextran, into single cells in discrete regions of the embryo and tracing the labeled cell's fate over time3-5. While effective, these methods are technically demanding and require specialized equipment not commonly found in zebrafish labs. Recently, photoconvertable fluorescent proteins, such as Eos and Kaede, which irreversibly switch from green to red fluorescence when exposed to ultraviolet light, are seeing increased use in zebrafish6-8. The optical clarity of the zebrafish embryo and the relative ease of transgenesis have made these particularily attractive tools for lineage labeling and to observe the migration of cells in vivo7. Despite their utility, these proteins have some disadvantages compared to dye-mediated lineage labeling methods. The most crucial is the difficulty we have found in obtaining high 3-D resolution during photoconversion of these proteins. In this light, perhaps the best combination of resolution and ease of use for lineage labeling in zebrafish makes use of caged fluorescein dextran, a fluorescent dye that is bound to a quenching group that masks its fluorescence9. The dye can then be "uncaged" (released from the quenching group) within a specific cell using UV light from a laser or mercury lamp, allowing visualization of its fluorescence or immunodetection. Unlike iontophoretic methods, caged fluorescein can be injected with standard injection apparatuses and uncaged with an epifluorescence microscope equipped with a pinhole10. In addition, antibodies against fluorescein detect only the uncaged form, and the epitope survives fixation well11. Finally, caged fluorescein can be activated with very high 3-D resolution, especially if two-photon microscopy is employed 12,13. This protocol describes a method of lineage labeling by caged fluorescein and laser uncaging. Subsequenctly, uncaged fluorescein is detected simultaneously with other epitopes such as GFP by labeling with antibodies.

This protocol delineates a way to label and trace the fate of small groups of cells zebrafish embryos using UV-uncaging of caged fluorescein, followed by whole mount immunolabeling to amplify the signal from the uncaged fluorescein.

Lineage תיוג של תאים דג הזברה עם לייזר Uncagable והעמסת dextran

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

אינדוקציה וניתוח של אפיתל לmesenchymal מעבר

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

Nephrotoxin Microinjection דג הזברה למודל אי-ספיקת כליות חריפה

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

ממשק משתמש גרפי למעקב בסיוע תוכנה של ריכוז חלבון בהפרעות ניידות דינמיות

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal muscle development and regeneration, and the autophagic process has been described in several muscular pathologies and age-related muscle disorders. A recently described block of the autophagic process that correlates with the functional exhaustion of satellite cells during muscle repair supports the notion that active autophagy is coupled with productive muscle regeneration. These data uncover the crucial role of autophagy in satellite cell activation during muscle regeneration in both normal and pathological conditions, such as muscular dystrophies. Here, we provide a protocol to monitor the autophagic process in the adult Muscle Stem Cell (MuSC) compartment during muscle regenerative conditions. This protocol describes the setup methodology to perform in situ immunofluorescence imaging of LC3, an autophagy marker, and MyoD, a myogenic lineage marker, in muscle tissue sections from control and injured mice. The methodology reported allows for monitoring the autophagic process in one specific cell compartment, the MuSC compartment, which plays a central role in orchestrating muscle regeneration.

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Active autophagy is associated with productive muscle regeneration, which is essential for Muscle Stem Cell (MuSC) activation. Here, we provide a protocol for the in situ detection of LC3, an autophagy marker in MyoD-positive MuSCs of muscle tissue sections from control and injured mice.

ב Situ Immunofluorescent מכתים של autophagy בתאי גזע שריר

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal ...