עבודה זו מומנה על ידי מענק NIH Ey10540 ללטף.

1. איריס תרבית תאים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> איסוף העיניים של 7 סלמנדרות (הרדים ב 0.1% אתיל חומצה 3-aminobenzoate מתאן sulfonic שהוכנו PBS) וכן מקום מגנזיום סידן חופשי הנקס פתרון (CMF). </li><li style=";text-align:right;direction:rtl"> שנה כפפות עבודה למכסה המנוע בתרבית רקמה. </li><li style=";text-align:right;direction:rtl"> לעקר כדורי העין Lugol&#39;s-EtOH למשך 3 שניות. </li><li style=";text-align:right;direction:rtl"> לשטוף 2 פעמים ב CMF. </li><li style=";text-align:right;direction:rtl"> העברת עיניים הנקס ולהתחיל לנתיחה. </li><li style=";text-align:right;direction:rtl"> לנתח אירוס-הקרנית מורכבת להסיר הרשתית העצבית. </li><li style=";text-align:right;direction:rtl"> העברת מתחמי לתוך תבשיל חדש של הנקס. </li><li style=";text-align:right;direction:rtl"> באמצעות # 15 אזמל, מתחמי נפרד לחצאים הגבי ו הגחון. </li><li style=";text-align:right;direction:rtl"> הסר שברי קשתית (הגחון הראשונה) ומניחים בצלחת המכילה 1 מ&quot;ל L-15. </li><li style=";text-align:right;direction:rtl"> הוסף 15% (150 UL) נפח dispase (7.5 יחידות / ריכוז מ&quot;ל). </li><li style=";text-align:right;direction:rtl"> לדגור על 27 מעלות צלזיוס למשך 2 שעות (צלחת לעטוף עם parafilm). </li><li style=";text-align:right;direction:rtl"> לבודד תאים IPE מ stroma (טריפסין מקום פתרון בשעה 27 ° C). </li><li style=";text-align:right;direction:rtl"> איסוף תאים IPE לתוך צינור 1.5 מ&quot;ל. </li><li style=";text-align:right;direction:rtl"> צנטריפוגה בסל&quot;ד 1000 ב RT (טמפרטורת החדר) למשך 2 דקות, להסיר supernatant. </li><li style=";text-align:right;direction:rtl"> לשטוף עם 1 מ&quot;ל הנקס צנטריפוגות ב 1000 סל&quot;ד ב RT במשך 2 דקות, להסיר supernatant. </li><li style=";text-align:right;direction:rtl"> הוסף 1 מ&quot;ל פתרון טריפסין, דגירה עבור 2 שעות 27 ° C. </li><li style=";text-align:right;direction:rtl"> צנטריפוגה ב 1000 סל&quot;ד ב RT במשך 2 דקות, להסיר טריפסין. </li><li style=";text-align:right;direction:rtl"> הוסף 1 מ&quot;ל L-15 לשטוף, צנטריפוגה בסל&quot;ד 1000 למשך 2 דקות. </li><li style=";text-align:right;direction:rtl"> הוסף ul 800 L-15, pipet לאט למעלה ולמטה ~ 10 פעמים (יותר מ 12 פעמים מקטין הדבקות). </li><li style=";text-align:right;direction:rtl"> פלייט התאים IPE בצלחת 24 קולגן גם לדגור מצופה על 27 מעלות צלזיוס למשך 2 שבועות (בדרך כלל, 4 הגבי ו 4 בארות הגחון מתקבלים 7 סלמנדרות). </li><li style=";text-align:right;direction:rtl"> בשלב זה תאים מתאימים transfection גן או טיפול עם גורמים כדי לקבוע את תפקידם או גרימת הפרעה התחדשות העדשה. </li></ol> 2. צבירה של תאים איריס <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> כדי צלחות המכיל תאים איריס להוסיף 20μl של פתרון dispase עד בינוני 400 מערבולת μl, בעדינות. </li><li style=";text-align:right;direction:rtl"> דגירה צלחות על 27 מעלות צלזיוס למשך לילה. </li><li style=";text-align:right;direction:rtl"> Pipet בעדינות מעלה ומטה כדי לסלק תאים </li><li style=";text-align:right;direction:rtl"> המקום התאים לתוך צינור Eppendorf ספין 2 דקות בסל&quot;ד 1000 ב RT. </li><li style=";text-align:right;direction:rtl"> הסר בינוני לשטוף ידי הוספת L-15 שלם 1 מ&quot;ל, ספין 2 דקות בסל&quot;ד 1000 ב RT, להסיר בינוני. </li><li style=";text-align:right;direction:rtl"> השתמש 2000-7000 התאים לכל המצרפי. הוסף ul 200 L-15 לכל המצרפי לתוך צינור אחד. </li><li style=";text-align:right;direction:rtl"> פיצול תאים (200 μl) לתוך צינורות חדשים Eppendorf. </li><li style=";text-align:right;direction:rtl"> ספין בסל&quot;ד 1000 דקות 2 ב RT. </li><li style=";text-align:right;direction:rtl"> דגירה של 48 שעות ב 27 ° C. </li></ol> 3. השרשה של תאים Aggregrated <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ביצוע חתך בקרנית העין סלמנדרה ולהסיר את העדשה. </li><li style=";text-align:right;direction:rtl"> לאחר הסרת העדשות, המקום המצרפי תא IPE ממש מתחת רקמת הקרנית בצד הגחוני של העין. </li><li style=";text-align:right;direction:rtl"> סלמנדרות נשמרים למשך חודש כדי לאפשר להם לחדש את העדשה. </li></ol> 4. והטבעה של העין ניוט <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הסר את העיניים סלמנדרה מושתל עם במצטבר ולמקם אותו פוספט בופר סליין (PBS). </li><li style=";text-align:right;direction:rtl"> תקן ב paraformaldehyde 4% ב 4 ° C למשך לפחות 4 שעות או למשך הלילה. </li><li style=";text-align:right;direction:rtl"> שטפי ב-PBS, 4 ° C למשך 30 דקות. </li><li style=";text-align:right;direction:rtl"> פנקו את זה עם מלח 0.85%: 4 ° C, 30 דקות. </li><li style=";text-align:right;direction:rtl"> להתרחץ מלוחים / אתנול (1:1): RT במשך 15 דקות. </li><li style=";text-align:right;direction:rtl"> לשטוף באתנול 70%: RT, 15 דקות. חזור על זה פעם. </li><li style=";text-align:right;direction:rtl"> לשטוף באתנול 85% אתנול ו 95% ב RT, 30 דקות כל אחד </li><li style=";text-align:right;direction:rtl"> שטפי ב 100% אתנול: RT, 30 דקות. חזור על זה פעם. </li><li style=";text-align:right;direction:rtl"> חנות ב 4 מעלות צלזיוס או להמשיך. </li><li style=";text-align:right;direction:rtl"> שטפי ב קסילן 100%: RT, 30 דקות. חזור על זה פעם. </li><li style=";text-align:right;direction:rtl"> פנקו עם קסילן / פרפין (1:1): 60 ° C, 45 דקות. </li><li style=";text-align:right;direction:rtl"> פנקו עם פרפין 100%: 60 ° C, 20 דקות. חזור על זה עוד פעמיים. </li><li style=";text-align:right;direction:rtl"> שבץ הטבעה תבניות. </li></ol> 5. חתך <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכן 15 מיקרומטר חלקים של העין מוטבע באמצעות microtome. </li><li style=";text-align:right;direction:rtl"> מניחים את הסעיפים עין בשקופית מצופה ג&#39;לטין ולהשאיר אותה בשקופית חם. </li></ol> 6. הכתמה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> המקום שקופיות קסילן במשך 10 דקות. חזור על זה עוד פעם. </li><li style=";text-align:right;direction:rtl"> מימה ב: 100%, 95%, 80%, 70%, אתנול 30% עבור כל 1 דקה </li><li style=";text-align:right;direction:rtl"> לשטוף במים DI לרגע. </li><li style=";text-align:right;direction:rtl"> שטפי ב PBS במשך 15 דקות. </li><li style=";text-align:right;direction:rtl"> שטפי ב PBST (0.2% טריטון X-100 ב PBS) במשך 15 דקות. </li><li style=";text-align:right;direction:rtl"> שטפי ב PBS במשך 15 דקות. </li><li style=";text-align:right;direction:rtl"> מקום חסימת פתרון (נוגדן עיזים 10% משני) במשך שעה. </li><li style=";text-align:right;direction:rtl"> מקום הנוגדן העיקרי בין לילה. </li><li style=";text-align:right;direction:rtl"> לשטוף נוגדן ראשוני PBS במשך 15 דקות. </li><li style=";text-align:right;direction:rtl"> שטפי ב PBST במשך 15 דקות. </li><li style=";text-align:right;direction:rtl"> שטפי ב PBS במשך 15 דקות. </li><li style=";text-align:right;direction:rtl"> מקום נוגדנים משני 2 שעות בחושך (1:100 דילול ב PBS). </li><li style=";text-align:right;direction:rtl"> שטפי ב-PBS, PBST, ו pbs במשך 15 דקות כל אחד, ואז הר. </li><li style=";text-align:right;direction:rtl"> שים את השקופיות תחת מיקרוסקופ פלואורסצנטי. </li></ol> 7. נציג תוצאות: הליך זה של culturing סלמנדרה irתאים נוצל כדי לחקור את פוטנציאל התחדשות של הגבי לבין תאים IPE הגחון. יתר על כן, אפשר גם ללמוד גנים מסוימים שתורמים כלפי מנגנון התחדשות העדשה בעין סלמנדרה. כאשר תאים בתרבית כבר 2 שבועות (איור 1) הם יכולים להיות transfected ידי הגנים לבחון את תפקידם של התחדשות העדשה. עניין מיוחד הם גנים שעשויים לגרום הקשתית הגחון. מאז תאים הגחון קשתית לא יכול transdifferentiate העדשה (איור 2) הפונקציה אינדוקטיבי של הגן מועמד ניתן ללמוד. בעבר, השימוש בטכניקה זו הראינו כי כאשר שש 3 באה לידי ביטוי על פני בנוכחות אינדוקציה החומצה הרטינואית העדשה נצפתה מן הגחון איריס 6. באיור 3 ניתן לראות כי במצטבר הגחון איריס הולידה עדשה בוגרת הבדיל (ראש חץ), לא שונה מאשר עדשות המארח של איריס הגבי (חץ). <img src="/files/ftp_upload/2713/2713fig1.jpg" alt="איור 1" /> באיור 1. א) תאים הגב איריס פיגמנט אפיתל בתרבית במבחנה לתקופה של 2 שבועות. ב) הגחון התאים בקשתית פיגמנט אפיתל בתרבית במבחנה לתקופה של 2 שבועות. שים לב פיגמנטציה נמשכת עד לשלב זה הן הגבי ו הגחון התאים בקשתית. <img src="/files/ftp_upload/2713/2713fig2.jpg" alt="איור 2" /> איור 2. התחדשות היכולת של התאים בתרבית IPE. א) עדשה אינדוקציה מן המצרפי הגבי תא IPE (ראש חץ). מארח העדשה אינדוקציה מן איריס הגבי (חץ), די: איריס הגבי, vi: איריס הגחון, le: אפיתל העדשה, אם: סיבי העדשה. ב) העדר אינדוקציה העדשה המצרפי הגחון תא IPE (ראש חץ). מארח העדשה אינדוקציה מן איריס הגבי (חץ). <img src="/files/ftp_upload/2713/2713fig3.jpg" alt="איור 3" /> איור 3. אינדוקציה עדשה מ המצרפי הגחון transfected עם שישה-3 ו שטופלו החומצה הרטינואית. מארח העדשה אינדוקציה מן איריס הגבי (חץ). עדשה אינדוקציה מן המצרפי הגחוני (ראש חץ).

<ol>
	<li>Tsonis, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+in+vertebrates.">Regeneration in vertebrates.</a> Dev Biol. 221, 273-284 (2000).</li><li>Tsonis, P. A., Madhavan, M., Tancous, E. E., Del Rio-Tsonis, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+newt's+eye+view+of+lens+regeneration.">A newt's eye view of lens regeneration.</a> Int. J. Dev. Biol. 48, 975-980 (2004).</li><li>Del Rio-Tsonis, K., Tsonis, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eye+regeneration+at+the+molecular+age.">Eye regeneration at the molecular age.</a> Dev. Dyn. , 226-2211 (2003).</li><li>Okamoto, M., Ito, M., Owaribe, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Difference+between+dorsal+and+ventral+iris+in+lens+producing+potency+in+normal+lens+regeneration+is+maintained+after+dissociation+and+reaggregation+of+cells+from+the+adult+newt,+Cynops+pyrrhogaster.">Difference between dorsal and ventral iris in lens producing potency in normal lens regeneration is maintained after dissociation and reaggregation of cells from the adult newt, Cynops pyrrhogaster.</a> Develop Growth Differ. 40, 11-18 (1998).</li><li>Hayashi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+transfection+system+for+functional+gene+analysis+in+adult+amphibian+lens+regeneration.">Highly efficient transfection system for functional gene analysis in adult amphibian lens regeneration.</a> Develop Growth Differ. 43, 361-370 (2001).</li><li>Grogg, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BMP+inhibition-driven+regulation+of+six-3+underlies+induction+of+newt+lens+regeneration.">BMP inhibition-driven regulation of six-3 underlies induction of newt lens regeneration.</a> Nature. 438, 858-862 (2005).</li><li>Nakamura, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miRNAs+in+Newt+Lens+Regeneration:+Specific+Control+of+Proliferation+and+Evidence+for+miRNA+Networking.">miRNAs in Newt Lens Regeneration: Specific Control of Proliferation and Evidence for miRNA Networking.</a> PLoS One. 5, e12058-e12058 (2010).</li><li>Madhavan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+Pax-6+in+lens+regeneration.">The role of Pax-6 in lens regeneration.</a> Proc. Natl. Acad. Sci. U.S.A. 103, 14848-14853 (2006).</li><li>Maki, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oocyte-type+linker+histone+B4+is+required+for+transdifferentiation+of+somatic+cells+in+vivo.">Oocyte-type linker histone B4 is required for transdifferentiation of somatic cells in vivo.</a> FASEB J. 24, 3462-3467 (2010).</li></ol>

אין ניגודי אינטרסים הכריז.

פרוטוקול זה הקימה במערכת במבחנה ללמוד התחדשות מנגנוני העדשה סלמנדרות. מאז אגרגטים (הגבי או הגחון פעל בנאמנות שלהם בהתנהגות vivo במהלך רגנרציה בטכניקה זו יכולה להקל על מאמץ אדיר נדרש transgenesis ב סלמנדרות והוא יכול לשמש למטרות רווח של הפונקציה, כמו גם אובדן של ניסויים פונקציה 7,8,9. כמו כן , אגרגטים או אירוסים בכללותו יכול בקלות להיות מטופלים עם גורמי גדילה ולבחון את השפעתם כמתואר בפרוטוקול זה. לדוגמה את התפקיד של BMP מסלול נחקרה באמצעות טכניקה זו 6.

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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<tr>
<td>Lugol's solution</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>L-6146</td>
<td>&nbsp;</td>
<tr>
<td>HEPES</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H-4034</td>
<td>&nbsp;</td>
<tr>
<td>Ethyl 3-aminobenzoate methanesulfonic acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>E-10521</td>
<td>&nbsp;</td>
<tr>
<td>Dispase</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>17105-041</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin 1:250</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>27250018</td>
<td>&nbsp;</td>
<tr>
<td>L-15 ( Leibovitz)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>L-4386</td>
<td>&nbsp;</td>
<tr>
<td>DNase 1</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D5025-150KU</td>
<td>&nbsp;</td>
<tr>
<td>Kanamycin Sulfate</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>100x, 15160</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>F4135</td>
<td>&nbsp;</td>
<tr>
<td>Fungizone (amphotericin B solution)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A2942</td>
<td>&nbsp;</td>
<tr>
<td>24 well collagen coated plate</td>
<td>&nbsp;</td>
<td>BD biosciences</td>
<td>354408</td>
<td>&nbsp;</td>		</tbody>
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a system for culturing iris pigment epithelial cells to study lens regeneration in newt

סלמנדרות כמו סלמנדרה ו האמביסטומה להחזיק את היכולת להתחדש רבים של חלקים שאבדו שלה הגוף כגון גפיים, זנב עם חוט השדרה עיניים, מוח, לב, הלסת 1. באופן ספציפי, סלמנדרות ייחודיים ביכולתה העדשה התחדשות. לאחר הסרת העדשות, תאים IPE של הקשתית הגב transdifferentiate לתאי העדשה ולבסוף טופס עדשה חדשה בעוד כחודש 2,3. מאפיין זה של התחדשות לעולם לא הוצגו על ידי התאים הגחון איריס. פוטנציאל התחדשות של תאים קשתית ניתן ללמוד על ידי ביצוע השתלות של תאים במבחנה IPE תרבותי. עבור התרבות, התאים הגבי ו הגחון איריס מבודדים הראשון בעין ותרבותי בנפרד עבור פרק זמן של שבועות 2 (איור 1). תאים אלה בתרבית הם reaggregated ו מושתל חזרה העין סלמנדרה. מחקרים שנעשו בעבר הראו כי reaggregate הגבי שומרת יכולת להרכיב עדשות שלה בעוד המצרפי הגחון אינו טופס עדשה, השחזור, וכך בתהליך vivo (איור 2) 4,5. שיטה זו של קביעת פוטנציאל התחדשות של תאים הגבי ו הגחון איריס שימושית מאוד ללמוד את התפקיד של גנים וחלבונים המעורבים התחדשות העדשה.

Watch this Scientific Journal Video about A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt at JoVE.com

A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt

ב סלמנדרה, העדשה מחדש תמיד מתוך איריס הגבי על ידי transdifferentiation של תאים פיגמנט אפיתל קשתית (IPEs). כאן אנו מתארים את הליך הגבי ו הגחון התרבות תאים סלמנדרה פטרוכימיים ההשתלה שלהם לעין סלמנדרה. התאים המושתלים נלמדים מכן על ידי חתך רקמות אימונוהיסטוכימיה.

מערכת culturing איריס תאים פיגמנט אפיתל לחקר התחדשות עדשה של ניוט

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, ...

a-system-for-culturing-iris-pigment-epithelial-cells-to-study-lens

Research

JoVE Journal

Biology

11.7K Views.  University of Dayton. ב סלמנדרה, העדשה מחדש תמיד מתוך איריס הגבי על ידי transdifferentiation של תאים פיגמנט אפיתל קשתית (IPEs). כאן אנו מתארים את הליך הגבי ו הגחון התרבות תאים סלמנדרה פטרוכימיים ההשתלה שלהם לעין סלמנדרה. התאים המושתלים נלמדים מכן על ידי חתך רקמות אימונוהיסטוכימיה.

Video: A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt

Laser Ablation of the Zebrafish Pronephros to Study Renal Epithelial Regeneration

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in renal failure1. AKI can be caused by a number of factors including ischemia, drug-based toxicity, or obstructive injury1. This results in an inability to maintain fluid and electrolyte homeostasis. While AKI has been observed for decades, effective clinical therapies have yet to be developed. Intriguingly, some patients with AKI recover renal functions over time, a mysterious phenomenon that has been only rudimentally characterized1,2. Research using mammalian models of AKI has shown that ischemic or nephrotoxin-injured kidneys experience epithelial cell death in nephron tubules1,2, the functional units of the kidney that are made up of a series of specialized regions (segments) of epithelial cell types3. Within nephrons, epithelial cell death is highest in proximal tubule cells. There is evidence that suggests cell destruction is followed by dedifferentiation, proliferation, and migration of surrounding epithelial cells, which can regenerate the nephron entirely1,2. However, there are many unanswered questions about the mechanisms of renal epithelial regeneration, ranging from the signals that modulate these events to reasons for the wide variation of abilities among humans to regenerate injured kidneys.
The larval zebrafish provides an excellent model to study kidney epithelial regeneration as its pronephric kidney is comprised of nephrons that are conserved with higher vertebrates including mammals4,5. The nephrons of zebrafish larvae can be visualized with fluorescence techniques because of the relative transparency of the young zebrafish6. This provides a unique opportunity to image cell and molecular changes in real-time, in contrast to mammalian models where nephrons are inaccessible because the kidneys are structurally complex systems internalized within the animal. Recent studies have employed the aminoglycoside gentamicin as a toxic causative agent for study of AKI and subsequent renal failure: gentamicin and other antibiotics have been shown to cause AKI in humans, and researchers have formulated methods to use this agent to trigger kidney damage in zebrafish7,8. However, the effects of aminoglycoside toxicity in zebrafish larvae are catastrophic and lethal, which presents a difficulty when studying epithelial regeneration and function over time. Our method presents the use of targeted cell ablation as a novel tool for the study of epithelial injury in zebrafish. Laser ablation gives researchers the ability to induce cell death in a limited population of cells. Varying areas of cells can be targeted based on morphological location, function, or even expression of a particular cellular phenotype. Thus, laser ablation will increase the specificity of what researchers can study, and can be a powerful new approach to shed light on the mechanisms of renal epithelial regeneration. This protocol can be broadly applied to target cell populations in other organs in the zebrafish embryo to study injury and regeneration in any number of contexts of interest.

Acute kidney injury (AKI) in humans is a common clinical problem caused by damage to the epithelial cells that comprise kidney nephrons, and AKI is associated with high mortality rates of 50-70%1. Following epithelial cell destruction, nephrons have a limited ability to regenerate, though the mechanisms and limitations that guide this phenomenon remain poorly understood. In this video article, we describe our technique for targeted laser ablation of kidney nephron cells in the zebrafish embryo kidney, or pronephros. Our new method can be used to complement nephrotoxicity-induced models of AKI and gain a high-resolution understanding of the cell and molecular alterations that are associated with epithelial regeneration in the kidney nephron.

אבלציה בלייזר של דג הזברה כלית ראשית לחקר התחדשות אפיתל הכליה

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in ...

A 3D System for Culturing Human Articular Chondrocytes in Synovial Fluid

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not regenerate very efficiently in vivo; and as a result, osteoarthritis leads to irreversible cartilage loss and is accompanied by chronic pain and immobility 1,2. Cartilage tissue engineering offers promising potential to regenerate and restore tissue function. This technology typically involves seeding chondrocytes into natural or synthetic scaffolds and culturing the resulting 3D construct in a balanced medium over a period of time with a goal of engineering a biochemically and biomechanically mature tissue that can be transplanted into a defect site in vivo 3-6. Achieving an optimal condition for chondrocyte growth and matrix deposition is essential for the success of cartilage tissue engineering. 
 In the native joint cavity, cartilage at the articular surface of the bone is bathed in synovial fluid. This clear and viscous fluid provides nutrients to the avascular articular cartilage and contains growth factors, cytokines and enzymes that are important for chondrocyte metabolism 7,8. Furthermore, synovial fluid facilitates low-friction movement between cartilaginous surfaces mainly through secreting two key components, hyaluronan and lubricin 9 10. In contrast, tissue engineered cartilage is most often cultured in artificial media. While these media are likely able to provide more defined conditions for studying chondrocyte metabolism, synovial fluid most accurately reflects the natural environment of which articular chondrocytes reside in.
 Indeed, synovial fluid has the advantage of being easy to obtain and store, and can often be regularly replenished by the body. Several groups have supplemented the culture medium with synovial fluid in growing human, bovine, rabbit and dog chondrocytes, but mostly used only low levels of synovial fluid (below 20&#37;) 11-25. While chicken, horse and human chondrocytes have been cultured in the medium with higher percentage of synovial fluid, these culture systems were two-dimensional 26-28. Here we present our method of culturing human articular chondrocytes in a 3D system with a high percentage of synovial fluid (up to 100&#37;) over a period of 21 days. In doing so, we overcame a major hurdle presented by the high viscosity of the synovial fluid. This system provides the possibility of studying human chondrocytes in synovial fluid in a 3D setting, which can be further combined with two other important factors (oxygen tension and mechanical loading) 29,30 that constitute the natural environment for cartilage to mimic the natural milieu for cartilage growth. Furthermore, This system may also be used for assaying synovial fluid activity on chondrocytes and provide a platform for developing cartilage regeneration technologies and therapeutic options for arthritis.

A 3D system of culturing human articular chondrocytes in high levels of synovial fluid is described. Synovial fluid reflects the most natural microenvironment for articular cartilage, and can be easily obtained and stored. This system thus can be used for studying cartilage regeneration and for screening therapeutics for treating arthritis.

מערכת 3D עבור chondrocytes culturing האדם במפרק של נוזל סינוביאלי

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

עשה זאת בעצמך התקן להבראת דוגמאות Cryopreserved בטעות ירד לתוך טנקים אחסון קריוגני

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

ניתוח גנטי מהיר של איתות אפיתל-mesenchymal במהלך התחדשות שיער

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

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 ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. 
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. 
In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

Culturing של אדם תאי אפיתל באף בממשק האוויר הנוזלי

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

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 ...

Intestinal Epithelial Regeneration in Response to Ionizing Irradiation

The intestinal epithelium consists of a single layer of cells yet contains multiple types of terminally differentiated cells, which are generated by the active proliferation of intestinal stem cells located at the bottom of intestinal crypts. However, during events of acute intestinal injury, these active intestinal stem cells undergo cell death. Gamma irradiation is a widely used colorectal cancer treatment, which, while therapeutically efficacious, has the side effect of depleting the active stem cell pool. Indeed, patients frequently experience gastrointestinal radiation syndrome while undergoing radiotherapy, in part due to active stem cell depletion. The loss of active intestinal stem cells in intestinal crypts activates a pool of typically quiescent reserve intestinal stem cells and induces dedifferentiation of secretory and enterocyte precursor cells. If not for these cells, the intestinal epithelium would lack the ability to recover from radiotherapy and other such major tissue insults. New advances in lineage-tracing technologies allow tracking of the activation, differentiation, and migration of cells during regeneration and have been successfully employed for studying this in the gut. This study aims to depict a method for the analysis of cells within the mouse intestinal epithelium following radiation injury.

The gastrointestinal tract is one of the most sensitive organs to injury upon radiotherapeutic cancer treatments. It is simultaneously an organ system with one of the highest regenerative capacities following such insults. The presented protocol describes an efficient method to study the regenerative capacity of the intestinal epithelium.

התחדשות אפיתל המעי בתגובה לקרינה מייננת

The intestinal epithelium consists of a single layer of cells yet contains multiple types of terminally differentiated cells, which are generated by the ...