עבודה זו נתמכת על ידי מענקים מהבריאות האוסטרלית הלאומיות והמועצה למחקר רפואי (NHMRC # 559007) וסוכרת נעורי Research Foundation (JDRF 4-2006-1025) ותכנית התשתיות התפעולית של הממשלה ויקטוריאני. אנו מודים לחברי צוות טום מנדל איילט להשתלות תכנית איילט הבידוד למתן רקמות האדם. רקמות אדם נאספו ושימוש באישור אתי מקומי (בית חולי סנט וינסנט HREC-הבריאות של סנט וינסנט 011/04 וHREC-135/08).

1. הכנת רקמת טחול <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ההערה, כל החומר האנושי יש להתייחס כאל פוטנציאל מדבק וכל ההליכים צריכים להתנהל בקבינט זרימה למינרית שנייה ייצוגי. בעזרת מספריים ומלקחיים סטריליים, הסרת רקמת שומן וסיבית מסעיפי טחול (~ 1-2 סנטימטר בגודל) וחתוך אותו במידה של חומר הכמוסה החיצוני ככל האפשר. </li><li style=";text-align:right;direction:rtl"> לחתוך חתיכה קטנה (1-2 סנטימטר 3) של רקמת טחול ולמקם את כל חתיכה לתוך צינור סטרילי 50 מ&quot;ל פלקון. </li><li style=";text-align:right;direction:rtl"> Snap-להקפיא את החתיכות של רקמות על ידי טבילה בנוזל N 2. </li><li style=";text-align:right;direction:rtl"> חנות ב -80 ° C. פרוטוקול דומה מתאים לרקמות אחרות (הים). </li></ol> 2. הכנת איילט אנוש לאחסון <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> איוני תרבות בתקשורת CMRL. איסוף איונים בצינור תחתון 10 מיליליטר חרוטים ולשטוף פעמים ב PBS על ידי צנטריפוגה בסל&quot;ד 1500 למשך 5 דקות. יוצק את PBS ולנקז את החיץ שיורית על ידי נחת הצינור ההפוך בקצרה על מגבת נייר. היזהר שלאכדי לסלק את האיים. </li><li style=";text-align:right;direction:rtl"> ברגע שמרוקן, מחדש כובע צינור ונצמד הקפאה בחנקן נוזלי ובחנות -80 ° C. </li></ol> 3. הכנת תמצית <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכן Baw תמהיל (10:30:60% v / v) וחנות ב 4 ° C. </li><li style=";text-align:right;direction:rtl"> הסר את הצינור מ-80 ° C. להפשיר בטמפרטורת חדר. </li><li style=";text-align:right;direction:rtl"> הוסף Baw מספיק קרה כקרח כדי לכסות את פיסת הרקמה. לאיים להשתמש 3-5 מ&quot;ל. לרקמת טחול להשתמש 10-20 מ&quot;ל תלוי בגודל של פיסת הרקמה. </li><li style=";text-align:right;direction:rtl"> להרכיב homogenizer הרקמות. נקה ידי &#39;מאחד&#39; 10-20 מ&quot;ל של אתנול 70% / מים. </li><li style=";text-align:right;direction:rtl"> הומוגני הרקמה בצרורות מרובות, לאחר ביצוע בדיקת homogenizer לתוך הצינור עם פתרון Baw רקמות ו. לשמור על הצינור בדלי קרח לאחר מכן. </li><li style=";text-align:right;direction:rtl"> לנקות ביסודיות homogenizer בין דגימות, על ידי מאחד 10-20 מ&quot;ל של אתנול 70% / מים ולאחר מכן חיץ Baw. זה מונע זיהום צולב של רקמה בין samples.Dismantleand נקי עם אתנול 70% / מים אחרינודואר. </li><li style=";text-align:right;direction:rtl"> אם תמצית מכילה חומר מסיס רק נדרשה, חובצה את תמצית רקמה הומוגני ב4000 סל&quot;ד בRT למשך 10 דקות. אם תמצית גולמית יותר נדרשת, להסתובב בסל&quot;ד 1000 בRT למשך 5 דקות. הטכניקה המתאימה ביותר לחילוץ החלבונים Baw המסיס תלויה בניתוח במורד הזרם של החלבונים. לשימוש במבחנים אימונולוגיים פונקציונליים אנחנו היינו מציעים שננסה לפרק את החלק המסיס באוריאת ז 8 כפי שמצאנו בעבר זה להיות נסבל היטב 6. </li><li style=";text-align:right;direction:rtl"> העברת supernatant לצינור נקי ולשים אותו על קרח. </li><li style=";text-align:right;direction:rtl"> לקבוע את הריכוז של חלבון בתמצית באמצעות assay BCA או דומה. </li></ol> 4. הקפא תמציות ייבוש <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> בהתאם המסה של חלבון הנדרש (כלומר, 100 מיקרוגרם לצינור), לדלל את homogenate בהתאם ולוותר aliquots ל5.0 כותרת המ&quot;ל סטרילי, פלקון (12x75 מ&quot;מ) צינורות. לעתים קרובות אנו משתמשים ב100 מיקרוגרם / צינור. </li><li style=";text-align:right;direction:rtl"> השתמש 1מחט סטרילית 8-20 מד מזרק לעשות 3 חורים בכובע של כל צינור. </li><li style=";text-align:right;direction:rtl"> הקפא צינורות או על ידי הצבתן בקרח יבש ל~ 10 דקות או במקפיא -80 מעלות צלזיוס במשך&gt; 1 שעה. חנות ב -80 ° C עד מוכן לשים על lyophilizer. </li><li style=";text-align:right;direction:rtl"> לעבור על הקפאה יבשה יותר ומאפשר לאזן (-100 ° C). זה לוקח בערך 30 דקות. </li><li style=";text-align:right;direction:rtl"> בהתאם לנפח, ייבוש בהקפאה יכול להסתיים בתוך 3 שעות אבל באופן שגרתי להשאיר הדגימות שלנו בן לילה. </li><li style=";text-align:right;direction:rtl"> לאחר סיומו של מחזור הייבוש, לכבות משאבת ואקום ולאפשר לחץ לאט לאט אל תוך החדר. הסר מדף. </li><li style=";text-align:right;direction:rtl"> במכסת מנוע סטרילי, להסיר את המכסים המחוררים מהצינורות ולהחליף בחדשים (פלקון 352032). </li><li style=";text-align:right;direction:rtl"> צינורות חנות ב -20 ° C. דוגמאות ניתן לשחזר בתרבית, או חוצצים אחרים, ולהשתמש בפונקציה או מבחנים ביוכימיים. </li></ol> 5. נציג תוצאות איור 1 מציג את ההכתמה של חלבוןנטען ג&#39;ל עם תמצית מרקמת לבלב מדולדל איון (שכותרת acinar) טחול ומטוהרים ואיי איוני אדם (שכותרת). התוצאות מראות ייצוג טוב של חלבונים בעלי משקל מולקולרי שונה לכל רקמות. הקיבולת של תמציות רקמה כדי לעורר שגשוג תאי T אנושי נבדקה באמצעות assay CFSE מבוסס התפשטות 7 (איור 2). PBMC משמש assay זה בודד מאדם עם סוכרת מסוג 1. עוצמת התגובה מתבטאת כיחס בין מספר תאים עמומים CFSE ל5000 CD4 +, תאי CFSE בהירים ללא אנטיגן: תאים עמומים CFSE ל5000 CD4 +, תאים בהירים CFSE עם האנטיגן מדגימות שלושה עותקים 7. התוצאות מראות חלשה, אבל, ריבוי זיהוי בתגובה לacinar (CD1 = 3.5) ותגובה חזקה לתמצית איון (6.8). נגיף שפעת מומת (CDI = 142.6) כלול כביקורת חיובית. <img alt="איור 1" src="/files/ftp_upload/4230/4230fig1.jpg" /> איור 1. ג&#39;ל חלבון. <img alt="איור 2" src="/files/ftp_upload/4230/4230fig2.jpg" /> איור 2. תוצאות מבדיקת תפוצה CFSE מבוססת נגד acinar ותמצית איון. <a href="http://www.jove.com/files/ftp_upload/4230/4230fig2large.jpg" target="_blank">לחצו כאן לצפייה בדמות גדולה.</a>

<ol>
	<li>Mannering, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+approaches+to+measuring+human+islet-antigen+specific+T+cell+function+in+type+1+diabetes.">Current approaches to measuring human islet-antigen specific T cell function in type 1 diabetes.</a> Clin. Exp. Immunol. 162, 197-209 (2010).</li><li>Beckhove, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+T+cell-based+identification+of+human+tumor+tissue+antigens+by+automated+two-dimensional+protein+fractionation.">Rapid T cell-based identification of human tumor tissue antigens by automated two-dimensional protein fractionation.</a> J. Clin. Invest. 120, 2230-2242 (2010).</li><li>Mannering, S. I., Brodnicki, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+insights+into+CD4++T-cell+specificity+and+function+in+Type+1+diabetes.">Recent insights into CD4+ T-cell specificity and function in Type 1 diabetes.</a> Expert Review of Clinical Immunology. 3, 557-564 (2007).</li><li>Mannering, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+A-chain+of+insulin+is+a+hot-spot+for+CD4++T+cell+epitopes+in+human+type+1+diabetes.">The A-chain of insulin is a hot-spot for CD4+ T cell epitopes in human type 1 diabetes.</a> Clin. Exp. Immunol. 156, 226-231 (2009).</li><li>Mannering, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+insulin+A-chain+epitope+recognized+by+human+T+cells+is+posttranslationally+modified.">The insulin A-chain epitope recognized by human T cells is posttranslationally modified.</a> J. Exp. Med. 202, 1191-1197 (2005).</li><li>Moon, H. C., Joffe, M., Thomas, H. E., Kay, T. W. H., Mannering, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+extracting+tissue+proteins+for+use+in+lymphocyte+function+assays.">A method for extracting tissue proteins for use in lymphocyte function assays.</a> Journal of Immunological Methods. 359, 56-60 (2010).</li><li>Mannering, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sensitive+method+for+detecting+proliferation+of+rare+autoantigen-specific+human+T+cells.">A sensitive method for detecting proliferation of rare autoantigen-specific human T cells.</a> J. Immunol. Methods. 283, 173-183 (2003).</li><li>Mannering, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD4++T+Cell+Proliferation+in+Response+to+GAD+and+Proinsulin+in+Healthy,+Pre-diabetic,+and+Diabetic+Donors.">CD4+ T Cell Proliferation in Response to GAD and Proinsulin in Healthy, Pre-diabetic, and Diabetic Donors.</a> Ann. N.Y. Acad. Sci. 1037, 16-21 (2004).</li><li>Mannering, S. I., Purcell, A. W., Honeyman, M. C., McCluskey, J., Harrison, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+T-cells+recognise+N-terminally+Fmoc-modified+peptide.">Human T-cells recognise N-terminally Fmoc-modified peptide.</a> Vaccine. 21, 3638-3646 (2003).</li><li>Peakman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+preparations+of+GAD65,+proinsulin,+and+the+islet+tyrosine+phosphatase+IA-2+for+use+in+detection+of+autoreactive+T-cells+in+type+1+diabetes:+report+of+phase+II+of+the+Second+International+Immunology+of+Diabetes+Society+Workshop+for+Standardization+of+T-cell+assays+in+type+1+diabetes.">Characterization of preparations of GAD65, proinsulin, and the islet tyrosine phosphatase IA-2 for use in detection of autoreactive T-cells in type 1 diabetes: report of phase II of the Second International Immunology of Diabetes Society Workshop for Standardization of T-cell assays in type 1 diabetes.</a> Diabetes. 50, 1749-1754 (2001).</li><li>Honey, K., Rudensky, A. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lysosomal+cysteine+proteases+regulate+antigen+presentation.">Lysosomal cysteine proteases regulate antigen presentation.</a> Nat. Rev. Immunol. 3, 472-482 (2003).</li></ol>

אין ניגודי האינטרסים הכריזו.

פרוטוקול זה פותח בגלל שרצינו להפיק תמצית מרקמה האנושית שהייתה חופשי מכימיקלים רעילים כגון חומרי ניקוי. באופן ספציפי יש לנו השתמשנו בו כדי להכין תמצית של רקמה האנושית שניתן להשתמש במבחנים של תפקוד מערכת חיסון של אדם במבחנה. תמציות שהוכנו באמצעות פרוטוקול זה באו?...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם המגיב </td><td> חברה </td><td> מספר קטלוגים </td><td> תגובות </td></tr><tr><td> 5 מ&quot;ל 12 75 צינורות קלקר סטריליים מ&quot;מ x </td><td> BD פלקון </td><td> 352054 </td><td></td></tr><tr><td> כובעים לצינורות צינורות קלקר (לעיל) </td><td> BD פלקון </td><td> 352032 </td><td></td></tr><tr><td> 50 מיליליטר צינורות סטריליים </td><td> הקיטון דיקינסון </td><td> 352070 </td><td></td></tr><tr><td> Acetonitrile </td><td> כימיקלי Mallinckradt </td><td> 2856-10 </td><td></td></tr><tr><td> Butan-1-ol </td><td> סיגמה אולדריץ </td><td> 537993-IL </td><td></td></tr><tr><td> Homogenizer: PRO200 </td><td> Bio-אסטרטגיה </td><td> 01-01200 </td><td> 10 המ&quot;מ x 115 גנרטור משונן </td></tr><tr><td> Lyophilizer </td><td></td><td></td><td> Virtis, Benchtop 4K </td></tr><tr><td> 18-20 מד מחט סטרילית </td><td> הקיטון דיקינסון </td><td> REF 302032 </td><td></td></tr><tr><td> בינוני CMRL-1066 </td><td> סיגמא </td><td> C0422 </td><td></td></tr><tr><td> PBS </td><td> סיגמא </td><td> D8537 </td><td></td></tr></tbody></table> טבלת 1. ריאגנטים וציוד ספציפיים.

extraction of tissue antigens for functional assays

רבים מיעדי אנטיגן של תגובה חיסונית אדפטיבית, המוכרים על ידי תאי T, B ולא הוגדרו 1. זה נכון במיוחד במחל אוטואימוניות וסרטן 2. המטרה שלנו היא לחקור את האנטיגנים מוכרים על ידי תאי T אנושיים בסוג המחלה אוטואימונית 1 הסוכרת 1,3,4,5. כדי לנתח את התגובות אנושיות תא T נגד רקמות שבו אנטיגנים המוכרים על ידי תאי T אינם מזוהים פתחנו שיטה לחילוץ אנטיגנים חלבון מרקמה אנושית בתבנית תואמת עם מבחנים תפקודיים 6. תגובות בעבר, תאים-T לתמציות רקמות unpurified לא ניתן היו למדוד, משום ששיטות החילוץ להניב lysate שהכיל חומרי ניקוי שהיו רעילים לתאי דם היקפי אדם mononuclear. כאן אנו מתארים פרוטוקול לחילוץ חלבונים מרקמות אדם בתבנית שאינה רעיל לתאי T אנושיים. רקמת homogenized בתערובת של, אצטוניטריל butan-1-ol וואטאה (Baw). ריכוז החלבון בתמצית הרקמה נמדד ומסה ידועה של חלבון aliquoted לתוך צינורות. לאחר חילוץ, הממסים האורגניים יוסרו על ידי lyophilization. תמציות רקמות Lyophilized ניתן לאחסן עד נדרשים. לשימוש במבחנים של תפקוד מערכת חיסוני, השעיה של תאי חיסון, בתקשורת התרבות המתאימה, ניתן להוסיף ישירות לתמצית lyophilized. ייצור ציטוקינים ושגשוג על ידי PBMC, בתגובה לתמציות שהוכנו באמצעות שיטה זו, נמדדו בקלות. לפיכך, השיטה שלנו מאפשרת הכנה המהירה של lysates רקמות האנושיים שיכול לשמש כמקור לאנטיגנים בניתוח של תגובות תא T. אנו ממליצים כי שיטה זו תאפשר הניתוח של תגובות חיסוניות מסתגלות לרקמות בהשתלה, סרטן והתפתחות מחלה.

Watch this Scientific Journal Video about Extraction of Tissue Antigens for Functional Assays at JoVE.com

Extraction of Tissue Antigens for Functional Assays

פרוטוקול פשוט להכנת תמציות של רקמה אנושית שתשמש כמקור לאנטיגנים במבחנים תפקודיים תא T מתואר. שיטה זו מאפשרת תגובות תא T לאנטיגנים המופקים מרקמות כדי להימדד<em&gt; במבחנה</em&gt;.

הפקת אנטיגנים רקמות למבחנים פונקציונליים

Many of the antigen targets of adaptive immune response, recognized by B and T cells, have not been defined 1. This is particularly true in autoimmune ...

extraction-of-tissue-antigens-for-functional-assays

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10.8K Views.  St. Vincent's Institute of Medical Research. פרוטוקול פשוט להכנת תמציות של רקמה אנושית שתשמש כמקור לאנטיגנים במבחנים תפקודיים תא T מתואר. שיטה זו מאפשרת תגובות תא T לאנטיגנים המופקים מרקמות כדי להימדד במבחנה.

Video: Extraction of Tissue Antigens for Functional Assays

Light/dark Transition Test for Mice

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, however, can be used to delete or manipulate a specific gene in mice. The influence of a given gene on a specific behavior can then be determined by conducting behavioral analyses of the mutant mice. As a test for behavioral phenotyping of mutant mice, the light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. The test is based on the natural aversion of mice to brightly illuminated areas and on their spontaneous exploratory behavior in novel environments. The test is sensitive to anxiolytic drug treatment. The apparatus consists of a dark chamber and a brightly illuminated chamber. Mice are allowed to move freely between the two chambers. The number of entries into the bright chamber and the duration of time spent there are indices of bright-space anxiety in mice. To obtain phenotyping results of a strain of mutant mice that can be readily reproduced and compared with those of other mutants, the behavioral test methods should be as identical as possible between laboratories. The procedural differences that exist between laboratories, however, make it difficult to replicate or compare the results among laboratories. Here, we present our protocol for the light/dark transition test as a movie so that the details of the protocol can be demonstrated. In our laboratory, we have assessed more than 60 strains of mutant mice using the protocol shown in the movie. Those data will be disclosed as a part of a public database that we are now constructing.

Visualization of the protocol will facilitate understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used across laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test.

The light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. Here, we present a movie that shows detailed procedures on how we conduct the test.

אור / חושך מעבר מבחן עכברים

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, ...

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

אקטין Co-Assay שקיעה, כי ניתוח של חלבון מחייב את ה-F-אקטין

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the lipidic cubic phase or in meso method. The method has been shown to be quite versatile in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and alpha-helical and beta-barrel proteins. Recent successes using in meso crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour.

Herein is described the procedure implemented in the Caffrey Membrane Structural and Functional Biology Group to set up manually crystallization trials of membrane proteins in lipidic mesophases.

גיבוש חלבונים בממברנה עבור קביעת מבנה באמצעות Mesophases Lipidic

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from these animals reform the original body plan following regeneration of missing body structures. For example if a 'trunk' fragment is cut from an intact worm, a new 'head' will regenerate anteriorly and a 'tail' will regenerate posteriorly restoring the original 'head-to-tail' polarity of body structures prior to amputation (Figure 1A).
Regeneration is driven by planarian stem cells, known as 'neoblasts' which differentiate into ~30 different cell types during normal body homeostasis and enforced tissue regeneration. This regenerative process is robust and easy to demonstrate. Owing to the dedication of several pioneering labs, many tools and functional genetic methods have now been optimized for this model system. Consequently, considerable recent progress has been made in understanding and manipulating the molecular events underpinning planarian developmental plasticity2-9.
The planarian model system will be of interest to a broad range of scientists. For neuroscientists, the model affords the opportunity to study the regeneration of an entire nervous system, rather than simply the regrowth/repair of single nerve cell process that typically are the focus of study in many established models. Planarians express a plethora of neurotransmitters10, represent an important system for studying evolution of the central nervous system11, 12 and have behavioral screening potential13, 14. 
Regenerative outcomes are amenable to manipulation by pharmacological and genetic apparoaches. For example, drugs can be screened for effects on regeneration simply by placing body fragments in drug-containing solutions at different time points after amputation. The role of individual genes can be studied using knockdown methods (in vivo RNAi), which can be achieved either through cycles of microinjection or by feeding bacterially-expressed dsRNA constructs8, 9, 15. Both approaches can produce visually striking phenotypes at high penetrance- for example, regeneration of bipolar animals16-21. To facilitate adoption of this model and implementation of such methods, we showcase in this video article protocols for pharmacological and genetic assays (in vivo RNAi by feeding) using the planarian Dugesia japonica.

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

An attractive model for studying stem cell differentiation within a live animal is the planarian flatworm. Regeneration is studied by simple amputation experiments that are easily performed in a basic laboratory and are amenable to pharmacological and genetic (in vivo RNAi) manipulation as detailed by protocols in this article.

מבחני גנטית תרופתי ו פונקציונלית לתפעל התחדשות של Planarian Japonica Dugesia</em

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from ...

Quantitative Comparison of cis-Regulatory Element (CRE) Activities in Transgenic Drosophila melanogaster

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE possesses an arrangement of binding sites for several transcription factor proteins that confer a regulatory logic specifying when, where, and at what level the regulated gene(s) is expressed. The full set of CREs within an animal genome encodes the organism&prime;s program for development1, and empirical as well as theoretical studies indicate that mutations in CREs played a prominent role in morphological evolution2-4. Moreover, human genome wide association studies indicate that genetic variation in CREs contribute substantially to phenotypic variation5,6. Thus, understanding regulatory logic and how mutations affect such logic is a central goal of genetics. 
Reporter transgenes provide a powerful method to study the in vivo function of CREs. Here a known or suspected CRE sequence is coupled to heterologous promoter and coding sequences for a reporter gene encoding an easily observable protein product. When a reporter transgene is inserted into a host organism, the CRE&prime;s activity becomes visible in the form of the encoded reporter protein. P-element mediated transgenesis in the fruit fly species Drosophila (D.) melanogaster7 has been used for decades to introduce reporter transgenes into this model organism, though the genomic placement of transgenes is random. Hence, reporter gene activity is strongly influenced by the local chromatin and gene environment, limiting CRE comparisons to being qualitative. In recent years, the phiC31 based integration system was adapted for use in D. melanogaster to insert transgenes into specific genome landing sites8-10. This capability has made the quantitative measurement of gene and, relevant here, CRE activity11-13 feasible. The production of transgenic fruit flies can be outsourced, including phiC31-based integration, eliminating the need to purchase expensive equipment and/or have proficiency at specialized transgene injection protocols. 
Here, we present a general protocol to quantitatively evaluate a CRE&prime;s activity, and show how this approach can be used to measure the effects of an introduced mutation on a CRE&prime;s activity and to compare the activities of orthologous CREs. Although the examples given are for a CRE active during fruit fly metamorphosis, the approach can be applied to other developmental stages, fruit fly species, or model organisms. Ultimately, a more widespread use of this approach to study CREs should advance an understanding of regulatory logic and how logic can vary and evolve.

Quantitative Comparison of cis-Regulatory Element CRE Activities in Transgenic Drosophila melanogaster

Phenotypic variation for traits can result from mutations in cis-regulatory element (CRE) sequences that control gene expression patterns. Methods derived for use in Drosophila melanogaster can quantitatively compare the levels of spatial and temporal patterns of gene expression mediated by modified or naturally occurring CRE variants.

השוואה כמותית של Cis-רגולטוריות (Cre) אלמנט פעילות טראנסגנטי תסיסנית melanogaster</em

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

שימוש ברובוט להתגבשות תפוקה גבוהה של חלבונים בממברנה בMesophases Lipidic

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

Small-scale Nuclear Extracts for Functional Assays of Gene-expression Machineries

A great deal of progress in understanding gene expression has been made using in vitro systems. For most studies, functional assays are carried out using extracts that are prepared in bulk from 10-50 or more liters of cells grown in suspension. However, these large-scale preparations are not amenable to rapidly testing in vitro effects that result from a variety of in vivo cellular treatments or conditions. This journal video article shows a method for preparing functional small-scale nuclear extracts, using HeLa cells as an example. This method is carried out using as few as three 150 mm plates of cells grown as adherent monolayers. To illustrate the efficiency of the small-scale extracts, we show that they are as active as bulk nuclear extracts for coupled RNA Polymerase II transcription/splicing reactions. To demonstrate the utility of the extract protocol, we show that splicing is abolished in extracts prepared from HeLa cells treated with the splicing inhibitor drug E7107. The small-scale protocol should be generally applicable to any process or cell type that can be investigated in vitro using cellular extracts. These include patient cells that are only available in limited quantities or cells exposed to numerous agents such as drugs, DNA damaging agents, RNAi, or transfection, which require the use of small cell populations. In addition, small amounts of freshly grown cells are convenient and/or required for some applications.

A protocol for preparation of robust, small-scale HeLa nuclear extracts is described. This protocol is valuable for assays that require use of small populations of cells, such as cells treated with drugs or RNAi. The method should be applicable to a wide variety of gene expression assays and other cell types, including patient cells.

בקנה מידה קטן תמציות גרעיני עבור מבחני פונקציונלית של הגן ביטוי Machineries

A great deal of progress in understanding gene expression has been made using in vitro systems.  For most studies, functional assays are carried out using ...

Viability Assays for Cells in Culture

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized viability assays are expensive in terms of equipment but can be faster and more objective than manual cell counts. The present report describes the use of three such viability assays. Two of these assays are infrared and one is luminescent. Both infrared assays rely on a 16&#160;bit Odyssey Imager. One infrared assay uses the DRAQ5 stain for nuclei combined with the Sapphire stain for cytosol and is visualized in the 700 nm channel. The other infrared assay, an In-Cell Western, uses antibodies against cytoskeletal proteins (&#945;-tubulin or microtubule associated protein 2) and labels them in the 800 nm channel. The third viability assay is a commonly used luminescent assay for ATP, but we use a quarter of the recommended volume to save on cost. These measurements are all linear and correlate with the number of cells plated, but vary in sensitivity. All three assays circumvent time-consuming microscopy and sample the entire well, thereby reducing sampling error. Finally, all of the assays can easily be completed within one day of the end of the experiment, allowing greater numbers of experiments to be performed within short timeframes. However, they all rely on the assumption that cell numbers remain in proportion to signal strength after treatments, an assumption that is sometimes not met, especially for cellular ATP. Furthermore, if cells increase or decrease in size after treatment, this might affect signal strength without affecting cell number. We conclude that all viability assays, including manual counts, suffer from a number of caveats, but that computerized viability assays are well worth the initial investment. Using all three assays together yields a comprehensive view of cellular structure and function.

Therapeutic compounds are often first examined in vitro with viability assays. Blind cell counts by a human observer can be highly sensitive to small changes in cell number but do not assess function. Computerized viability assays, as described here, can assess both structure and function in an objective manner.

מבחני כדאיות לתאים בתרבית

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

בידוד וניתוח פונקציונלי של המיטוכונדריה מתאים בתרבית ורקמות עכבר

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction

Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in molecular analysis is complicated both by heterogeneous tissue composition and low yields when extracting nucleic acids. A literature search revealed a paucity of protocols addressing these issues, and none that showed a validated method for simultaneous extraction of RNA and DNA from regions of interest in FFPET. This method addresses both issues. Tissue specificity was achieved by mapping cancer areas of interest on microscope slides and transferring annotations onto FFPET blocks. Tissue cores were harvested from areas of interest using 0.6 mm microarray punches. Nucleic acid extraction was performed using a commercial FFPET extraction system, with modifications to homogenization, deparaffinization, and Proteinase K digestion steps to improve tissue digestion and increase nucleic acid yields. The modified protocol yields sufficient quantity and quality of nucleic acids for use in a number of downstream analyses, including a multi-analyte gene expression platform, as well as reverse transcriptase coupled real time PCR analysis of mRNA expression, and methylation-specific PCR (MSP) analysis of DNA methylation.

This modified extraction protocol improves RNA and DNA yields from more precisely targeted regions of interest in histopathologic tissue blocks.

הכין ליבות רקמות פרפין מוטבע פורמלין-קבועות הוא רנ&quot;א הפקת DNA

Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in ...