עבודה זו נתמכה על ידי מ &#39;מרגרט Alkek קרן אל RHF.

חלק 1: ספסל להגדיר <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> חדר לח מוכן ידי הצבת Kimwipe / DDH 2 O בתא פלסטיק. </li><li style=";text-align:right;direction:rtl"> צינור פלקון לאסוף אלבומין, מנות לתרבות, טבעות, watchglass וסילוק פסולת ממוקמות על הספסל. </li><li style=";text-align:right;direction:rtl"> צלחת פיירקס מלא מלוחים 1.4 l (ראה הערות [א]). </li></ol> חלק 2: העובר explanted ב מלוחים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ביצים יוסרו מן החממה לאחר 16 שעות (שלב 4). ביצה היא opene ידי הקשה על פגז עם מלקחיים. פיסות מעטפת יוסרו. </li><li style=";text-align:right;direction:rtl"> אלבומין דק נאסף בצינור פלקון. אלבומין עבה יוסר עם מלקחיים. </li><li style=";text-align:right;direction:rtl"> העובר ממוקם צלחת פלסטיק בתוך צלחת מלוחים. אלבומין הנותרים יוסר עם מלקחיים. </li></ol> חלק 3: העובר במרכז הטבעת <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> שק חלמון מוטה עם מלקחיים כך העובר הפונה כלפי מעלה. </li><li style=";text-align:right;direction:rtl"> שק החלמון הוא חתך ברמה של קו המשווה או למטה. </li><li style=";text-align:right;direction:rtl"> בעזרת מלקחיים בסדר, קרום vitelline הוא קילף במהירות. הממברנה vitelline מכוונת כך לצד גרגרי שלה (shinny הלא) הפנים כלפי מעלה. </li><li style=";text-align:right;direction:rtl"> בעזרת מלקחיים בסדר, קרום vitelline מושם על זכוכית השעון. </li><li style=";text-align:right;direction:rtl"> בעזרת מלקחיים בסדר, טבעת זכוכית מוחל על גבי קרום vitelline ואת העובר במרכז. </li><li style=";text-align:right;direction:rtl"> הממברנה vitelline הוא הרים סביב הקצוות של טבעת הזכוכית. האסיפה יוסר המנה מלוחים. </li></ol> חלק 4: תרבות מוגדרת תחת מיקרוסקופ <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הממברנה vitelline יוסר מעל טבעת הזכוכית באמצעות מלקחיים קנס תחת מיקרוסקופ. </li><li style=";text-align:right;direction:rtl"> בעזרת פיפטה פסטר, מלוחים נמחקת בקצה החיצוני של הטבעת. </li><li style=";text-align:right;direction:rtl"> בעזרת מספריים בסדר, עודף קרום vitelline מוסר מהקצה הפנימי של טבעת הזכוכית. הטיפול נלקח לא לחדור קרום עם פיפטה או מלקחיים. </li><li style=";text-align:right;direction:rtl"> העובר היא שטפה בעדינות עם מלח כדי להסיר את ממברנות התאים רופף חלמון. </li><li style=";text-align:right;direction:rtl"> 200 μl מלוחים מתווסף הקצה החיצוני של טבעת (זה יקל על העברת מאוחר יותר). </li><li style=";text-align:right;direction:rtl"> הרכבה מכוסה עם צלחת פלסטיק הפוכה. </li></ol> חלק 5: תרבות מועבר חממה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> צלחת תרבות פלקון מסומן. </li><li style=";text-align:right;direction:rtl"> 2.5ml של אלבומין דק נוסף בתחתית המנה פלקון. </li><li style=";text-align:right;direction:rtl"> 200μl של אלבומין דק מתווסף לקצה הפנימי של המכסה של המנה פלקון. </li><li style=";text-align:right;direction:rtl"> צלחת הפוכה יוסר הרכבה. </li><li style=";text-align:right;direction:rtl"> בעזרת מלקחיים בסדר, טבעת הזכוכית slided בשולי זכוכית השעון, והועבר המנה פלקון. </li><li style=";text-align:right;direction:rtl"> מלוחים כל שנותר הוא להסיר את המשטח הפנימי של הטבעת. </li><li style=";text-align:right;direction:rtl"> המנה פלקון מכוסה במכסה וחתום. </li><li style=";text-align:right;direction:rtl"> הרכבה ממוקם בתא לח ואז בחממה. </li><li style=";text-align:right;direction:rtl"> הם עוברים בתרבית עד 24 שעות ב 38 ° C. </li></ol> חלק 6: בעקבות התרבות, העובר הוא קבוע; תרבות מועבר חממה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> צלחת קיבוע מלא קר כקרח PBS (או depc-PBS אם הם עוברים להיות מעובד שכן הכלאה באתרו). </li><li style=";text-align:right;direction:rtl"> התרבות היא להסיר את האינקובטור והניח מיד על הקרח. המנה תרבות מלא מיד עם קרים כקרח PBS / depc-PBS. </li><li style=";text-align:right;direction:rtl"> העובר מנותקת הממברנה vitelline. העובר מועבר המנה קיבעון, באמצעות הקצה הקהה של פיפטה פסטר. </li><li style=";text-align:right;direction:rtl"> העובר הוא מרותקים באמצעות בוטה, הסתיים מלקחיים בסדר. PBS מכסה את המנה קיבעון מוסר. מיצב נוסף (ראה הערות [b]). </li><li style=";text-align:right;direction:rtl"> בהתאם ליישום, העובר הוא קבוע עבור שעות 6-8 ב 4 ° C (cryostat), O / N ב 4 ° C (ב מנח), או 1 שעות ב RT עבור אימונוהיסטוכימיה הר שלם. </li><li style=";text-align:right;direction:rtl"> מקבע היא להסיר והחליפו עם קרים כקרח PBS / depc PBS. </li><li style=";text-align:right;direction:rtl"> עבור יישומים במורד הזרם כמו הכלאה באתרו או מכתים immantibody: מערכת העצבים והלב הם מחורר באמצעות מחט סוף בוטה microcapillary או סכין microdissection, זה ימנע השמנה של בדיקה / נוגדן צעדים מאוחר יותר. הערות: [א] מלוחים כולל: פתרון (עבור l 1): 121.0 גרם NaCl, KCl 15.5 גרם, 10.4 גרם CaCl 2 .2 H 2 O, 12.7 גרם MgCl 2 0.6 H 2 O; פתרון B (עבור l 1) : 2.4 גרם Na 2 HPO 4 .2 H 2 O, 0.2 גרם לאא 2 PO 4 .2 H 2 O; החיטוי, לפני השימוש, לערבב 120 מ&quot;ל עם 2700 מ&quot;ל H 2 O: הוסף 180 מ&quot;ל ב מיקס [1]; [b] מקבע מוכן רק לפני השימוש (4% PFA ב PBS או depc-PBS על מנח ב). </li></ol> חלק 7: תוצאות נציג לפני התרבות, העובר נמצא בשלב פרימיטיבי פס (HH4). בסוף התקופה, תרבות, העובר התפתח HH10 (אורך 2-3 מ&quot;מ) גלוי במרכז צלחת את התרבות. אפשר תווית קבוצת תאים עם carbocyanine DiI פלורסנטבדיוק לפני התרבות (0h) ופעל תנועתם לאורך כל התקופה והתרבות. במקרה זה, התאים מתחת הצומת של Hensen (ח&quot;נ) תויגו עם DiI. תאים אלה מוצגים לתרום somites בהדרגה מתפתחות (סום) ו notochord (n). <img src="/files/ftp_upload/old/903_2008_10_20_2/903_Figure-01.png" alt="" width="780" height="228" /> חלק 8: פתרון בעיות <table style="width: 698px; height: 241px;;text-align:right;direction:rtl" border="1" cellspacing="0" cellpadding="0" width="698"><tbody><tr><th> בעיה </th><th> לגרום </th><th> תרופה </th></tr><tr><td> העובר נשאר עם חלמון במקום יורד עם קרום (שלב 2) </td><td> מסכן איכות הביצית </td><td> בקש מיוצר טרי ביצים, ביצים חנות ב 13 ° C עם קבלת. דגירה הביצים יום זהה לתאריך ההגעה. </td></tr><tr><td> Vitelline שקופיות קרום מן המיקום של שען הבאים כוס טבעת (שלב 3) </td><td> אלבומין שרידי </td><td> בשלב 2, לוודא שכל אלבומין יוסר ידי משיכתו הרחק קרום עם מלקחיים מוטה </td></tr><tr><td> העובר אינו נגיש: טמון מתחת הממברנה (שלב 4) </td><td> מהצד הלא נכון של הממברנה הוא כלפי מעלה </td><td> בשלב 3, יש לוודא את הצד של הקרום המכיל גרגרי חלמון פונה מעלה. הצד המבריק, מלוטש צריך הפנים כלפי מטה. </td></tr><tr><td> העובר שקועים בתרבות הבאים מלוחים / אלבומין (שלב 6) </td><td> חור בקרום; מלוחים / אלבומין שמאל בתוך טבעת לפני התרבות </td><td> בשלב 5, לוודא שכל אלבומין / מלוחים יוסר בתוך הטבעת, בשלב 4, הקפד לא לחדור קרום עם מלקחיים (להשתמש במלקחיים בוטה הסתיים) </td></tr><tr><td> העובר התפרקה בעקבות תרבות </td><td> זיהום בקטריאלי </td><td> לעקר את כל כלי וכלי זכוכית, שימוש באנטיביוטיקה / antimycotic </td></tr></tbody></table>

<ol>
	<li>Pannett, P. A., Compton, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cultivation+of+tissues+in+saline.">The cultivation of tissues in saline.</a> Lancet. 206, 381-384 (1924).</li><li>New, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+technique+for+the+cultivation+of+the+chick+embryo+in+vitro.">A new technique for the cultivation of the chick embryo in vitro.</a> J. Embryol. Exp. Morph. 3, 326-331 (1955).</li><li>Alvarez, I. S., Araujo, M., Nieto, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+induction+in+whole+chick+embryo+cultures+by+FGF.">Neural induction in whole chick embryo cultures by FGF.</a> Dev. Biol. 199, 42-54 (1998).</li><li>Psychoyos, D., Stern, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restoration+of+the+organizer+after+radical+ablation+of+Hensen's+node+and+the+anterior+primitive+streak+in+the+chick+embryo.">Restoration of the organizer after radical ablation of Hensen's node and the anterior primitive streak in the chick embryo.</a> Development. 122, 3263-3273 (1996).</li><li>Psychoyos, D., Stern, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fates+and+migratory+routes+of+primitive+streak+cells+in+the+chick+embryo.">Fates and migratory routes of primitive streak cells in the chick embryo.</a> Development. 122, 1523-1534 (1996).</li><li>Voiculescu, O., Papanayotou, C., Stern, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatially+and+temporally+controlled+electroporation+of+early+chick+embryos.">Spatially and temporally controlled electroporation of early chick embryos.</a> Nature Protoc. 3, 419-426 (2008).</li></ol>

השיטה תרבות חדשה 2 יכולים לשמש למגוון רחב של יישומים, החל שתלי של גורם גדילה המכיל חרוזים 3, אל הר שלמה הכלאה באתרו כל אימונוהיסטוכימיה הר 4. התרבות במשך 24 שעות מאפשרת ניטור רציף של ההתפתחות העוברית ביישומים כגון ניתוח זמן לשגות תנועת התא 5 או ניטו?...

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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<tr>
<td>Eggs</td>
<td>Animal</td>
<td>Charles River Laboratories</td>
<td>Premium Fertile</td>
<td>&nbsp;</td>
<tr>
<td>Stereomicroscope</td>
<td>Microscope</td>
<td>Leica Microsystems</td>
<td>MZ9.5 or similar</td>
<td>&nbsp;</td>
<tr>
<td>Marsh Automatic Incubator</td>
<td>Tool</td>
<td>Lyon</td>
<td>RX</td>
<td>&nbsp;</td>
<tr>
<td>Hybridization Incubator</td>
<td>Tool</td>
<td>Robbins Scientific</td>
<td>M1000</td>
<td>&nbsp;</td>
<tr>
<td>Pyrex dish (2)</td>
<td>Tool</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Watchmaker&#x2019;s glass 50mm</td>
<td>Tool</td>
<td>VWR</td>
<td>66112-060</td>
<td>&nbsp;</td>
<tr>
<td>Glass rings</td>
<td>Tool</td>
<td>Physical Plant facility</td>
<td>&nbsp;</td>
<td>cut 4 mm thick sections of glass tubing (27 mm outer diam, 25 mm inner diam). Do not fine polish.</td>
</tr>
<tr>
<td>Curved Forceps (1)</td>
<td>Surgery</td>
<td>Electron Microscopy Sciences</td>
<td>72991-4C</td>
<td>&nbsp;</td>
<tr>
<td>Forceps (2)</td>
<td>Surgery</td>
<td>Fine Science Tools</td>
<td>11002-13</td>
<td>blunt ended using sharpening Stone and 100ul mineral oil</td>
</tr>
<tr>
<td>Sharpening Stone Dan&#x2019;s Black Arkansas</td>
<td>Surgery</td>
<td>Electron Microscopy Sciences</td>
<td>62082-00</td>
<td>&nbsp;</td>
<tr>
<td>Fine scissors</td>
<td>Surgery</td>
<td>Fine Science Tools</td>
<td>14161-10</td>
<td>&nbsp;</td>
<tr>
<td>Plastic dishes</td>
<td>Tool</td>
<td>Falcon</td>
<td>353001</td>
<td>&nbsp;</td>
<tr>
<td>Rubber Bulb</td>
<td>Tool</td>
<td>Electron Microscopy Sciences</td>
<td>70980</td>
<td>&nbsp;</td>
<tr>
<td>Pasteur Capillary Pipette</td>
<td>Tool</td>
<td>Electron Microscopy Sciences</td>
<td>70950-12</td>
<td>round edge under flame</td>
</tr>
<tr>
<td>Microcapillary tube</td>
<td>Surgery</td>
<td>Sigma</td>
<td>P1049-1PAK</td>
<td>Pull using vertical micropipette puller; blunt end with fine forceps</td>
</tr>
<tr>
<td>Microdissecting knife</td>
<td>Surgery</td>
<td>Fine Science Tools</td>
<td>10056-12</td>
<td>Use to puncture cavities prior to in situ hybridization</td>
</tr>
<tr>
<td>Minuten pins 0.2mm diam</td>
<td>Surgery</td>
<td>Fine Science Tools</td>
<td>26002-20</td>
<td>&nbsp;</td>
<tr>
<td>Sylgard 184 Silicon Elastomer Curing Agent and Base</td>
<td>Reagent</td>
<td>Dow Corning</td>
<td>0001986475</td>
<td>Mix 1 part Curing Agent, 9 parts Base; set O/N at 37C</td>
</tr>
<tr>
<td>Diethylpyrocarbonate (depc)</td>
<td>Reagent</td>
<td>Acros Organics</td>
<td>10025025</td>
<td>Add 1ml depc to 1l PBS; shake; autoclave</td>
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method for culture of early chick embryos ex vivo (new culture)

עובר אפרוח הוא כלי רב ערך במחקר ההתפתחות העוברית המוקדמת. שקיפות, שלה נגישות וקלות מניפולציה, ולהפוך אותו כלי אידיאלי לחקר היווצרות של דפוסים, somite המוח, בצינור העצבי ואת primordia לב. יישומים של התרבות עובר אפרוח כוללים electroporation של בונה DNA או RNA על מנת לנתח את תפקיד הגן, שתלי של גורם גדילה חרוזים מצופים כגון FGFs ו BMPs, כמו גם הר שלם הכלאה אימונוהיסטוכימיה באתרו. סרט הווידאו הזה מדגים את השלבים השונים בתרבות עובר אפרוח: ראשית, העובר בתוך explanted מלוחים. ואז, העובר מתמקדת טבעת זכוכית. קרום המקיף את העובר הם הרים לאורך קירות של הטבעת. הטבעת ממוקמת כך בצלחת תרבות המכיל מאגר של albumine. המנה תרבות אטום והניחו בתא לח, שבו העובר הוא תרבותי של עד 24 שעות. לבסוף, העובר יוסר הטבעת, קבוע ומעובדים עבור יישומים נוספים. מדריך לפתרון בעיות מוצג גם.

Watch this Scientific Journal Video about Method for Culture of Early Chick Embryos ex vivo New Culture at JoVE.com

Method for Culture of Early Chick Embryos ex vivo New Culture

וידאו זה מדגים תרבות חדש, שיטה שבאמצעותה הם עוברים חומוס בתרבית מחוץ הביצה עד 24 שעות. שיטה זו מאפשרת ללמוד פיתוח מוקדם (פס פרימיטיביים עד 14 סום.), תקופה המקבילה E7-9 בעכבר. יישומים של טכניקה זו כוללים electroporation, ב הכלאה אימונוהיסטוכימיה באתרו.

שיטת לתרבות מוקדם של עוברים צ&#39;יק לשעבר vivo (תרבות חדש)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

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Method for Culture of Early Chick Embryos ex vivo (New Culture)

15.5K Views.  Institute of Biosciences and Technology - Texas A&M Health Science Center. וידאו זה מדגים תרבות חדש, שיטה שבאמצעותה הם עוברים חומוס בתרבית מחוץ הביצה עד 24 שעות. שיטה זו מאפשרת ללמוד פיתוח מוקדם (פס פרימיטיביים עד 14 סום.), תקופה המקבילה E7-9 בעכבר. יישומים של טכניקה זו כוללים electroporation, ב הכלאה אימונוהיסטוכימיה באתרו.

Video: Method for Culture of Early Chick Embryos ex vivo New Culture

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

הר שלם כפול באתרו הכלאה של עוברים צ&#39;יק מוקדם

Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study angiogenesis but also to study tumorigenesis. After the chick chorioallantoic membrane (CAM) has developed, its blood vessel network can be easily accessed, manipulated and observed and therefore provides an optimal setting for angiogenesis assays. Since the lymphoid system is not fully developed until late stages of incubation, the chick embryo serves as a naturally immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition to nurturing developing allo- and xenografts, the CAM blood vessel network provides a uniquely supportive environment for tumor cell intravasation, dissemination, and vascular arrest and a repository where arrested cells extravasate to form micro metastatic foci.
For experimental purposes, in most of the recent studies the CAM was exposed by cutting a window through the egg shell and experiments were carried out in ovo, resulting in significant limitations in the accessibility of the CAM and possibilities for observation and photo documentation of effects. When shell-less cultures of the chick embryo were used1-4, no experimental details were provided and, if published at all, the survival rates of these cultures were low. We refined the method of ex ovo culture of chick embryos significantly by introducing a rationally controlled extrusion of the egg content. These ex ovo cultures enhance the accessibility of the CAM and chick embryo, enabling easy in vivo documentation of effects and facilitating experimental manipulation of the embryo. This allows the successful application to a large number of scientific questions: (1) As an improved angiogenesis assay5,6, (2) an experimental set up for facilitated injections in the vitreous of the chick embryo eye7-9, (3) as a test environment for dissemination and intravasation of dispersed tumor cells from established cell lines inoculated on the CAM10-12, (4) as an improved sustaining system for successful transplantation and culture of limb buds of chicken and mice13 as well as (5) for grafting, propagation, and re-grafting of solid primary tumor tissue obtained from biopsies on the surface of the CAM14.
In this video article we describe the establishment of a refined chick ex ovo culture and CAM assay with survival rates over 50%. Besides we provide a step by step demonstration of the successful application of the ex ovo culture for a large number of scientific applications. 
Daniel S. Dohle, Susanne D. Pasa, and Sebastian Gustmann contributed equally to this study.

Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

The chick chorioallantoic membrane (CAM) is a unique, naturally immunodeficient supportive culture environment to study angiogenesis and tumorigenesis. This video article demonstrates the different steps in chick ex ovo culture, application of potentially angiogenic substances and successful inoculation of tumor cells and tissues on the surface of the CAM.

אפרוח אובו לשעבר תרבות אובו לשעבר CAM Assay: איך זה באמת עובד

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study ...

In vivo-like Organotypic Murine Retinal Wholemount Culture

Targeted ablations of genes and analysis of animal models is the classical strategy for enrolling specific retinal gene function. However, transgenic, retina-specific or conditional knockout mouse models often display early lethality or suffer from severe malformations, preventing an analysis beyond embryonic or early postnatal stages.
Primary cell culture is an alternative to investigate the effects of exogenously applied recombinant factors, overexpression of genes or siRNA-mediated gene knockdown in a controlled environment. Dissociated cell culture has the advantage that the endogenous signals reaching the target cells are reduced, thereby facilitating the identification of exogenously triggered effects after pharmacological manipulation. However, important cell-cell interactions are initially destroyed by enzymatic digestion or mechanical dissociation, even if re-aggregated retinospheroid cultures1 are used.
By contrast, organotypic retinal wholemount cultures provide a system close to the physiological in vivo situation with neuronal interactions and connections still preserved2-5.
In this video article we provide a step by step demonstration of (1) the establishment of in vivo-like organotypic retinal wholemount cultures including dissection peculiarities of embryonic, postnatal and adult murine eyes and (2) a dissociation and cytospin procedure for analysis of neuronal apoptosis and retinal cell proliferation in organotypic wholemounts, e.g. after culture in the presence of exogenously applied recombinant factors.

In vivo-like Organotypic Murine Retinal Wholemount Culture

This video article demonstrates the establishment of organotypic retinal wholemount cultures and a cytospin procedure for analysis of exogenously induced effects. Organotypic retinal wholemount cultures mimic the in vivo situation and significantly facilitate the accessibility of murine retinas for experimental manipulations while circumventing the disadvantages of classical murine animal models.

בשנת vivo, כמו תרבות Wholemount Murine Organotypic רשתית

Targeted ablations of genes and analysis of animal models is the classical strategy for enrolling specific retinal gene function. However, transgenic, ...

The Method of Rodent Whole Embryo Culture using the Rotator-type Bottle Culture System

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development and growth of mammalian embryos are critically dependent on the function of the placenta, WEC technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages during embryonic day (E) 6.5-E12.5 in the mouse or E8.5-E14.5 in the rat 2, 3, 4. In WEC, we can directly target desired areas of embryos using fine glass capillaries because embryos can be manipulated under the microscope. Therefore, rodent WEC is very useful technique when we want to study dynamic developmental processes of postimplanted mammalian embryos. Up to date, several types of WEC systems have been developed 1. Among those, the rotator-type bottle culture system is most popular and suitable for long-term culture of embryos at midgestation, i.e., after E9.5 and E11.5 in the mouse and rat, respectively 1. In this video protocol, we demonstrate our standard procedures of rat WEC after E12.5 using a refined model of the original rotator system, which was designed by New and Cockroft 5, 6, and introduce various applications of WEC technique for studies in mammalian developmental biology.

Whole embryo culture technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages. In this video protocol, we demonstrate our standard procedures of rat whole embryo culture after E12.5 using the rotator-type bottle culture system.

שיטת התרבות העובר שלמים מכרסמים באמצעות Rotator מסוג התרבות מערכת בקבוק

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development ...

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and uterine carcinomas, where death rates have fallen in recent years, ovarian cancer cure rates have remained relatively unchanged over the past two decades 1. This is largely due to the lack of appropriate screening tools for detection of early stage disease where surgery and chemotherapy are most effective 2, 3. As a result, most patients present with advanced stage disease and diffuse abdominal involvement. This is further complicated by the fact that ovarian cancer is a heterogeneous disease with multiple histologic subtypes 4, 5. Serous ovarian carcinoma (SOC) is the most common and aggressive subtype and the form most often associated with mutations in the BRCA genes. Current experimental models in this field involve the use of cancer cell lines and mouse models to better understand the initiating genetic events and pathogenesis of disease 6, 7. Recently, the fallopian tube has emerged as a novel site for the origin of SOC, with the fallopian tube (FT) secretory epithelial cell (FTSEC) as the proposed cell of origin 8, 9. There are currently no cell lines or culture systems available to study the FT epithelium or the FTSEC. Here we describe a novel ex vivo culture system where primary human FT epithelial cells are cultured in a manner that preserves their architecture, polarity, immunophenotype, and response to physiologic and genotoxic stressors. This ex vivo model provides a useful tool for the study of SOC, allowing a better understanding of how tumors can arise from this tissue, and the mechanisms involved in tumor initiation and progression.

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

The fallopian tube (FT) is emerging as an alternative site of origin for serous ovarian carcinoma (SOC). This protocol describes a novel method for the isolation and ex vivo culture of fallopian tube epithelial cells. This system recapitulates the in vivo epithelium and allows the study of SOC pathogenesis.

Ex Vivo התרבות של האדם תאים ראשוניים החצוצרה אפיתל

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and ...

Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. Tracking fiducial markers on the surfaces of these cellular sheets is a well-established method for estimating morphogenetic quantities such as growth, contraction, and shear. However, not all surface labeling techniques are readily adaptable to conventional imaging modalities and possess different advantages and limitations. Here, we describe two labeling methods and illustrate the utility of each technique. In the first method, hundreds of fluorescent labels are applied simultaneously to the embryo using magnetic iron particles. These labels are then used to quantity 2-D tissue deformations during morphogenesis. In the second method, polystyrene microspheres are used as contrast agents in non-invasive optical coherence tomography (OCT) imaging to track 3-D tissue deformations. These techniques have been successfully implemented in our lab to studythe physical mechanisms of early head fold, heart, and brain development, and should be adaptable to a wide range morphogenetic processes.

This article describes surface labeling and ex ovo tissue culture in the early chick embryo. Techniques amenable to time-lapse bright field, fluorescence, and optical coherence tomography imaging are presented. Tracking surface labels with high spatiotemporal resolution enables kinematic quantities such as morphogenetic strains (deformations) to be calculated in both two and three dimensions.

מעקב אחר דפורמציות המוךפו&quot;גנטי שהולך רקמות בעובר צ&#39;יק מוקדם

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. ...

Scale-Up of Mammalian Cell Culture using a New Multilayered Flask

A growing number of cell-based applications require large numbers of cells. Usage of single layer T-flasks, that are adequate during small-scale expansion, may become cumbersome, laborious and time-consuming when large numbers of cells are required. To address this need, the performance of a new multi-layered cell culture vessel to facilitate easy scale up of cells from single layered T-flasks will be discussed. The flasks tested are available in 3- and 5-layer format and enable culture and complete recovery of three and five times the number of cells respectively, compared to T-175 flasks. A key feature of the BD Multi-Flask is a mix/equilibration port that allows rapid in-vessel mixing as well as uniform distribution of cells and reagents within and 
between layers of each vessel and consistently produce cells that can be cultured in an environment that is congruent to T-175 flasks.
The design of these Multi-Flasks also allows for convenient pipette access for adding reagents and cells directly into the flasks as well as efficient recovery of valuable cells and reagents and reduces risk of contamination due to pouring. For applications where pouring is preferred over pipetting, the design allows for minimal residual liquid retention so as to reduce wastage of valuable cells and reagents.

Cells play an instrumental and increasing role in research, and the discovery and development of new therapeutics. With this increasing need for greater number of cells we need more efficient and effective ways for growing and harvesting attachment dependent cells. A Multilayered flask with the right features can serve this purpose.

Scale-Up של תרבית תאים היונקים באמצעות Flask multilayered חדש

A growing number of cell-based applications require large numbers of cells. Usage of single layer T-flasks, that are adequate during small-scale ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

רומן<em&gt; Ex vivo</em&gt; שיטת תרבות ללב העכבר העוברית

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...

The Slice Culture Method for Following Development of Tooth Germs In Explant Culture

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental biologist. For many organs it is difficult to access developing tissue to allow monitoring during ex vivo culture. The slice culture method allows access to tissue so that morphogenetic movements can be followed and specific cell populations can be targeted for manipulation or lineage tracing.
In this paper we describe a method of slice culture that has been very successful for culture of tooth germs in a range of species. The method provides excellent access to the tooth germs, which develop at a similar rate to that observed in vivo, surrounded by the other jaw tissues. This allows tissue interactions between the tooth and surrounding tissue to be monitored. Although this paper concentrates on tooth germs, the same protocol can be applied to follow development of a number of other organs, such as salivary glands, Meckel&#39;s cartilage, nasal glands, tongue, and ear.

Here we detail a method to culture tooth germs in mandible slices using a tissue chopper. This method allows unique access to the tooth during development, providing excellent opportunity for manipulation and lineage tracing, not available using more traditional culture methods.

פורסים תרבות השיטה למעקב אחר ההתפתחות של חיידקים בשיני explant תרבות

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental ...

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts migrate through the epidermis of the embryo where they subsequently colonize the developing hair follicles1,2. Neural crest cell migration is extensively studied in vitro but in vivo methods are still not well developed, especially in mammalian systems. One alternative is to use ex vivo organotypic culture3-6. Culture of mouse embryonic skin requires the maintenance of an air-liquid interface (ALI) across the surface of the tissue3,6. High resolution live-imaging of mouse embryonic skin has been hampered by the lack of a good method that not only maintains this ALI but also allows the culture to be inverted and therefore compatible with short working distance objective lenses and most confocal microscopes. This article describes recent improvements to a method that uses a gas permeable membrane to overcome these problems and allow high-resolution confocal imaging of embryonic skin in ex vivo culture6. By using a melanoblast specific Cre-recombinase expressing mouse line combined with the R26YFPR reporter line we are able to fluorescently label the melanoblast population within these skin cultures. The technique allows live-imaging of melanoblasts and observation of their behavior and interactions with the tissue in which they develop. Representative results are included to demonstrate the capability to live-image 6&#160;cultures in parallel.

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

We describe the dissection and ex vivo culture of mouse embryonic skin. The culture system maintains an air-liquid interface across the tissue surface and allows imaging on an inverted microscope. Melanoblasts, a component of the developing skin, are fluorescently labeled allowing their behavior to be observed using confocal microscopy.

אקס תרבות Vivo של עור העכבר עוברי וLive-הדמיה של הגירת Melanoblast

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts ...