ברצוננו להודות לרנה ל Pasker וHarpreet ק Brar לקבלת סיוע טכני מומחה לפרוטוקולים המתוארים בעבודה זו. יתר על כן, ברצוננו להכיר בחונכות של כריסטופר ב Newgard באוניברסיטת דיוק ואלן ד איתי באוניברסיטת ויסקונסין במדיסון, יחד עם התמיכה של חברי המעבדה שלהם, שאפשרו לנו את הזמן ואת התמיכה הנדרשת כדי למטב פרוטוקולים שתוארו. בפרט, אנו מודים לאנס Hohmeier, Danhong לו, והלנה וינפילד במעבדה Newgard ומרי Rabaglia במעבדה איתי לדיונים ועצות מועילים. עבודה זו נתמכה על ידי NIH מענק DK080845 וסוכרת נעורים 594 מענק קרן מחקר 17-2011-608 (לMEK)

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יש מחברים אין לחשוף.

עם השכיחות של הסוכרת צפויה להשפיע 7.7% מאוכלוסיית העולם, הדרישה של טכניקות מחקר רומן הכרחית לשניהם להבין ולטפל בסוכרת 18. בידוד איון הנוכחי הוא פרוטוקול מבוסס היטב המשמש לניסויים במבחנה וכבר הוצג בעבר עם 11,14,16 שינויים קלים. למרות שהפרשת אינסולין היא ייש...

התחזוקה הקפדנית של euglycemia היא הכרחית כדי למנוע נלוות כגון נוירופתיה, נפרופתיה ורטינופתיה, שהם כולם סימני ההיכר של פתולוגיה מסוג בלתי מבוקר 1 ו 2 סוכרת 1. פונקציה מופחתת β-תאים ומסה בשני מהסוג 1 וסוכרת 2 להפריע ריכוז הגלוקוז בדם 2. ואילו סוג החיסון בתיווך תוצאות 1 סוכרת מאובדן הרסני של β-תאים מייצרי אינסולין, הפרשה לקויה β-תאים לאינסולין ואינסולין איתות היקפית בסוכרת מסוג 2 יחד לקדם היפרגליקמיה, דיסליפידמיה, וייצור הגלוקוז בכבד מוגבר, שסופו של דבר תוצאה הוא גם אובדן מסת β-תא והפרשת אינסולין מקיבולת β-תאים בודדים 3. הבנת מנגנוני β-תאי הבסיס בהתקדמות מסוג 1 וסוכרת 2 יהיה בתקווה להצמיח טיפולים חדשניים למניעה וטיפול במחלות אלה. בti המבחנהמודלים תרבות ssue, כגון INS-1 וMIN6 הונצחו קווי β-תאים, יכולים להיות כלי שימושי להבנת פונקציות β-תאים ספציפיים. עם זאת, אינטראקציות בין סוגי התאים השונים בתוך איון עשויים עצמם מווסתים את תפקוד β-תאים. לדוגמא, ההשפעה אוטוקריני של גלוקגון (שוחררה מα-תאים) וסומטוסטטין (שוחרר מδ-תאים) בהגדלת והקטנת הפרשת אינסולין, בהתאמה, מדגים את החשיבות של תא תא קירבה בתגובה האנדוקרינית 4. יתר על כן, צמתים פער בין β-תאים להגביר את שחרור אינסולין 5. יתר על כן, למרות שצעדים שנעשו ביצירת קווי insulinoma שיותר טוב לשכפל את התגובה הפיזיולוגית של איים מבודדים לגלוקוז (לדוגמא, INS-1 נגזר 832/13 ו832/3 שורות תאים), היענות הגלוקוז שלהם עדיין שונה מעכברים רגילים האיונים 6,7. יתר על כן, התגובה של שורות תאים אלה משובטים insulinomaלאגוניסטים כמו גלוקגון פפטיד -1 (GLP-1) יכולה להיות שונה באופן דרמטי מזה, כמו גם מאיונים רגילים 6. לכן, שורות תאים הונצחו לא יכולות לייצג את המודל הטוב ביותר למנסה לאמוד סוכנים המשפיעים על ייצור cAMP. בניגוד לשורות תאי שמקורם בinsulinoma, לומד פונקצית β-תאים אך ורק במודלים של בעלי החיים כולו מציע סדרה של סיבוכים משלו. אחד האתגרים הגדולים ביותר בעבודה עם רקמה האנדוקרינית מודד את הריכוז המדויק של הורמון שפורסם. באופן ספציפי, הכבד ממלא תפקיד מרכזי בחילוף חומרים של אינסולין, וזרימת דם הלבלב הולכת ישירות אל הכבד. לפיכך, מדידת אינסולין בפלזמה לא יכולה לתאר במדויק את כמויות אינסולין שמופרשות מהלבלב עצמו או את ההשפעה של טיפולים שונים על שיעור הפרשת אינסולין 8. יתר על כן, חילוף חומרים של כליות של גלוקגון עלול להגביל את מהימנות פלט גלוקגון מהאיון α-תאים 9. לכן, בידוד איונים עכבר עיקריים לניסויים במבחנה מספק הבנה מדויקת יותר של אופן שבו איון מגיב לגירויים ספציפיים כדי להשלים את המדידות שנעשו בגוף חי. הפרוטוקול הנוכחי לבידוד של איי עכבר הוא פרוטוקול מבוסס היטב בשימוש על ידי מספר הקבוצות (עם שינויים קלים שעשויות לעזור כדי להגדיל את ההצלחה) 10,11. בנוסף, קביעת ייצור cAMP מאפשרת קריאה מתוך ישירה של היענות אינקרטין של β-התאים. בשילוב עם מדידת cAMP, תכולת חלבון והפרשת אינסולין גם ניתן לכמת מאותו מכין מדגם cAMP, עוזר לקבוע אם פגם בתפקודו β-תאים טמון פרוקסימלי או דיסטלי למחנה 10. התוכן הסופי cAMP ויישום הפרשת אינסולין בפרוטוקול זה יכול להיות כלי רב עוצמה להבנת ההשפעה של מרכיבי תרופות ומזון, בין השארים, על מחנה והפרשת אינסולין. בנוסף לגירוי מגלוקוז לבד, יכולה להיות בשימוש תרכובות אחרות כדי למדוד שינויים במחנה והפרשת אינסולין 10,11. לבסוף, למרות שהאינסולין הוא ההורמון העיקרי שאנחנו assay מאיים מבודדים, הורמונים אחרים, כגון גלוקגון וסומטוסטטין, כמו גם ציטוקינים, eicosanoids, וmonophosphate אדנוזין המחזורי, ניתן גם למדוד, או על ידי assay גירוי חולף או על ידי כימות הרמות שלהם במדיום תרבות 12. לבסוף, למרות שמחוץ לתחום של כתב היד הזה, בידוד איון עם שיטת בידוד collagenase תאר מאפשר לשימור איון, כך שיישומים רבים אחרים במורד הזרם עשויים להיות נרדפים, כגון השתלת איון, בידוד RNA בזמן אמת PCR כמוני או microarray מנתח, חלבון בידוד מערבי סופג, הטבעת איון וההדמיה immunofluorescent, ושילוב [3H]-תימידין כמדד לrepli תא איוןקטיון, שחלקם כבר תאר במאמרי יופיטר הקודמים 13-16. בסך הכל, בעקבות הליך בידוד איון שתואר בפרוטוקול עשוי לספק חוקר עם מידע חשוב ושימושי לפיתוח טיפולים וקידום גילוי תרופות שמטרתן שיפור תפקוד β-תאים.

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	<tbody>
		<tr height="64">
			<td height="64" style="height:64px;width:235px;">Collagenase: Collagenase from Clostridium histolyticum suitable for isolating active islets</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:136px;">C7657</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Ficoll 400</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td>F9378</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Hanks Balanced Salt Solution 10X</td>
			<td style="width:131px;">Invitrogen (Gibco)</td>
			<td>14065-056</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Hepes</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td>H3375</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">RPMI 1640 (powder)</td>
			<td style="width:131px;">Invitrogen (Gibco)</td>
			<td style="width:136px;">31800-022</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Albumin from Bovine Serum (BSA)</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td>A7888</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">3/0 Silk Suture Thread</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td>18020-30</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Dumont #5 Forceps</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td>11251-10</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">0.8 mm Forceps&#160;&#160;</td>
			<td style="width:131px;">Fine Science Tools&#160;&#160;</td>
			<td style="width:136px;">11050-10</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Curved Scissors</td>
			<td style="width:131px;">Fine Science Tools&#160;&#160;</td>
			<td style="width:136px;">14061-10</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:235px;">Vannas-T&#252;bingen Spring Scissors - Straight/Sharp/8.5 cm/5 mm Cutting Edge</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td style="width:136px;">15003-08</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Dissecting Scissors</td>
			<td style="width:131px;">Fine Science Tools&#160;&#160;</td>
			<td style="width:136px;">14002-14</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">5ml BD Luer-Lok Syringe</td>
			<td style="width:131px;">BD</td>
			<td style="width:136px;">309646</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">1ml BD syringe</td>
			<td style="width:131px;">BD</td>
			<td style="width:136px;">309628</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">30 G BD Needle 1/2&#34; Length</td>
			<td style="width:131px;">BD</td>
			<td style="width:136px;">305106</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">27 G BD Needle 1/2&#34; Length</td>
			<td style="width:131px;">BD</td>
			<td style="width:136px;">305109</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Sharpening Stone</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td style="width:136px;">29008-01</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">2-2-2 tribromoethanol</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:136px;">T48402-25G</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">2-methyl-2-butanol</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td>240486-100mL</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Sodium Chloride (NaCl)</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td>S9888</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Potassium Chloride</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td>P3911</td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:235px;">Monopotassium Phosphate (KH2PO4)</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:136px;">P0662</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:235px;">Sodium Bicarbonate (NaCHO3)</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:136px;">S6014</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:235px;">CaCl2 *2H2O</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:136px;">C3881</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:235px;">MgSO4 *7H2O</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:136px;">M9397</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Penicillin-Streptomycin</td>
			<td style="width:131px;">Invitrogen (Gibco)</td>
			<td style="width:136px;">15140-122</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:235px;">Heat Inactivated Fetal Bovine Serum (H.I. FBS)</td>
			<td style="width:131px;">Fisher Scientific</td>
			<td style="width:136px;">SH30088.03HI</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">3-Isobutyl-1-methylxanthine (IBMX)</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:136px;">5879-100MG</td>
		</tr>
	</tbody>
</table>

a method for mouse pancreatic islet isolation and intracellular camp determination

הגלוקוז לא מבוקר הוא סימן היכר של סוכרת ומקדם נלוות כמו נוירופתיה, נפרופתיה ורטינופתיה. עם השכיחות הגוברת של סוכרת, הן סוג החיסון בתיווך 1 וסוג 2 צמוד להשמנת יתר, מחקרים שמטרתם מגדירים הפתופיזיולוגיה סוכרת ומנגנונים טיפוליים הם בעלי חשיבות קריטית. Β-התאים של איי הלבלב לנגרהנס אחראים כראוי מפרישי אינסולין בתגובה לריכוז הגלוקוז בדם גבוה. בנוסף לגלוקוז וחומרים מזינים אחרים, β-התאים גם מגורה על ידי הורמונים מסוימים, כינה incretins, אשר מופרשים מהבטן בתגובה לארוחה ולפעול על קולטני β-תאים המגבירים את הייצור של monophosphate אדנוזין המחזורי תאיים ( cAMP). ירידה בתפקוד β-תא, מסה, ואת היענות אינקרטין הם הבינו היטב לתרום לפתופיזיולוגיה של סוכרת מסוג 2, וגם להיות מקושר יותר ויותר wiה סוכרת מסוג 1. הבידוד הנוכחי עכבר איון ופרוטוקול נחישות cAMP יכולים להיות כלי כדי לעזור להתוות מנגנוני קידום התקדמות מחלה והתערבויות טיפוליות, בעיקר אלה שמתווכים על ידי קולטנים אינקרטין או קשור קולטנים הפועלים באמצעות אפנון של ייצור cAMP תוך תאי. בעוד מדידות cAMP רק תתואר, פרוטוקול בידוד איון תאר יוצר הכנה נקייה שמאפשרת גם ליישומים רבים במורד הזרם אחרים, כוללים גלוקוז מגורה הפרשת אינסולין, [3 H]-תימידין התאגדות, שפע חלבון, וביטוי mRNA.

כדי להבטיח תשואת איון גבוהה במהלך בידוד, צריכים להיות אחרי הטכניקות ניתוחיות המפורטים בפרוטוקול באופן הדוק. למרות הטכניקות שהוצגו כאן תהיה מותאמות לכל מעבדה, יש כמה שלבים קריטיים שיובילו לבידוד מוצלח. על מנת להפוך את צינור המרה המשותף נגיש בקלות, מומלץ שהאיברים להיו...

Watch this Scientific Journal Video about A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination at JoVE.com

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

מנסה לאמוד בתפקוד β-תאי מבחנה באמצעות עכבר איים מבודדים לנגרהנס הוא מרכיב חשוב במחקר של הפתופיזיולוגיה סוכרת ותרופות. בעוד רבים יישומים במורד הזרם זמינים, פרוטוקול זה מתאר במפורש המדידה של monophosphate אדנוזין המחזורי תאיים (cAMP) כפרמטר חיוני קביעת פונקצית β-תאים.

שיטה לבידוד עכבר הלבלב איילט וקביעת תאית cAMP

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing ...

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65.1K Views.  University of Wisconsin-Madison.  מנסה לאמוד בתפקוד β-תאי מבחנה באמצעות עכבר איים מבודדים לנגרהנס הוא מרכיב חשוב במחקר של הפתופיזיולוגיה סוכרת ותרופות. בעוד רבים יישומים במורד הזרם זמינים, פרוטוקול זה מתאר במפורש המדידה של monophosphate אדנוזין המחזורי תאיים (cAMP) כפרמט...

Video: A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

Murine Pancreatic Islet Isolation

הלבלב Murine איילט בידוד

Human Pancreatic Islet Isolation: Part I: Digestion and Collection of Pancreatic Tissue

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of glucose monitoring and new insulin formulations, many individuals still develop devastating secondary complications. Pancreatic islet transplantation can restore near normal glucose control in diabetic patients 1, without the risk of serious hypoglycemic episodes that are associated with intensive insulin therapy. Providing sufficient islet mass is important for successful islet transplantation. However, donor characteristic, organ procurement and preservation affect the isolation outcome 2. At University of Illinois at Chicago (UIC) we have developed a successful isolation protocol with an improved purification gradient 3. The program started in January 2004, and more than 300 isolations were performed up to November 2008. The pancreata were sent in cold preservation solutions (UW, University of Wisconsin or HTK, Histidine-Tryptophan Ketoglutarate) 4-7 to the Cell Isolation Laboratory at UIC for islet isolation. Pancreatic islets were isolated using the UIC method, which is a modified version of the method originally described by Ricordi et al 8. Briefly, after cleaning the pancreas from the surrounding tissue, it was perfused with enzyme solution (Serva Collagenase + Neutral Protease or Sigma V enzyme). The distended pancreas was then transferred to the Ricordi digestion chamber, connected to a modified, closed circulation tubing system, and warmed up to 37&deg;C. During the digestion, the chamber was shaken gently. Samples were taken continuously to monitor the digestion progress. Once free islets were detected under the microscope, the digestion was stopped by flushing cold (4&deg;C) RPMI dilution solution (Mediatech, Herndon, VA) into the circulation system to dilute the enzyme. After being collected and washed in M199 media supplemented with human albumin, the tissue was sampled for pre-purification count and incubated with UW solution before purification. Purification process will be described in Part II: Purification and Culture of Human Islets.

Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part I: digestion and collection of pancreatic tissue) using a modified automated method.

בידוד האדם לבלב: אני חלק: העיכול אוסף של רקמת לבלב

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of ...

Human Pancreatic Islet Isolation: Part II: Purification and Culture of Human Islets

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of glucose monitoring and new insulin formulations, many individuals still develop devastating secondary complications. Pancreatic islet transplantation can restore near normal glucose control in diabetic patients 1, without the risk of serious hypoglycemic episodes that are associated with intensive insulin therapy. Providing sufficient islet mass is important for successful islet transplantation. However, donor characteristics, organ procurement and preservation affect the isolation outcome 2. At University of Illinois at Chicago (UIC) we developed a successful isolation protocol with an improved purification gradient 3. The program started in January 2004 and more than 300 isolations were performed up to November 2008. The pancreata were sent in cold preservation solutions (UW, University of Wisconsin or HTK, Histidine-Tryptophan Ketoglutarate) 4-7 to the Cell Isolation Laboratory at UIC for islet isolation. Pancreatic islets were isolated using the UIC method, which is a modified version of the method originally described by Ricordi et al 8. As described in Part I: Digestion and Collection of Pancreatic Tissue, human pancreas was trimmed, cannulated, perfused, and digested. After collection and at least 30 minutes of incubation in UW solution, the tissue was loaded in the cell separator (COBE 2991, Cobe, Lakewood, CO) for purification 3. Following purification, islet yield (expressed as islet equivalents, IEQ), tissue volume, and purity was determined according to standard methods 9. Isolated islets were cultured in CMRL-1066 media (Mediatech, Herndon, VA), supplemented with 1.5% human albumin, 0.1% insulin-transferrin-selenium (ITS), 1 ml of Ciprofloxacin, 5 ml o f 1M HEPES, and 14.5 ml of 7.5% Sodium Bicarbonate in T175 flasks at 37&deg;C overnight culture before islets were transplanted or used for research.

Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part II: purification and culture of human islets) using a modified automated method.

הלבלב האנושי בידוד איילט: חלק ב: טיהור והתרבות של איי האדם

Computer-assisted Large-scale Visualization and Quantification of Pancreatic Islet Mass, Size Distribution and Architecture

The pancreatic islet is a unique micro-organ composed of several hormone secreting endocrine cells such as beta-cells (insulin), alpha-cells (glucagon), and delta-cells (somatostatin) that are embedded in the exocrine tissues and comprise 1-2% of the entire pancreas. There is a close correlation between body and pancreas weight. Total beta-cell mass also increases proportionately to compensate for the demand for insulin in the body. What escapes this proportionate expansion is the size distribution of islets. Large animals such as humans share similar islet size distributions with mice, suggesting that this micro-organ has a certain size limit to be functional. The inability of large animal pancreata to generate proportionately larger islets is compensated for by an increase in the number of islets and by an increase in the proportion of larger islets in their overall islet size distribution. Furthermore, islets exhibit a striking plasticity in cellular composition and architecture among different species and also within the same species under various pathophysiological conditions. In the present study, we describe novel approaches for the analysis of biological image data in order to facilitate the automation of analytic processes, which allow for the analysis of large and heterogeneous data collections in the study of such dynamic biological processes and complex structures. Such studies have been hampered due to technical difficulties of unbiased sampling and generating large-scale data sets to precisely capture the complexity of biological processes of islet biology. Here we show methods to collect unbiased "representative" data within the limited availability of samples (or to minimize the sample collection) and the standard experimental settings, and to precisely analyze the complex three-dimensional structure of the islet. Computer-assisted automation allows for the collection and analysis of large-scale data sets and also assures unbiased interpretation of the data. Furthermore, the precise quantification of islet size distribution and spatial coordinates (i.e. X, Y, Z-positions) not only leads to an accurate visualization of pancreatic islet structure and composition, but also allows us to identify patterns during development and adaptation to altering conditions through mathematical modeling. The methods developed in this study are applicable to studies of many other systems and organisms as well.

Novel computer-assisted methods of large-scale procurement and analysis of immunohistochemically stained pancreatic specimens are described: (1) Virtual Slice capture of the entire section; (2) Mass analysis of large-scale data; (3) Reconstruction of 2D Virtual Slices; (4) 3D islet mapping; and (5) Mathematical analysis.

בסיוע מחשב ויזואליזציה בקנה מידה גדול כימות המוניים הלבלב איילט, התפלגות גודל ואדריכלות

The pancreatic islet is a unique micro-organ composed of several hormone secreting endocrine cells such as beta-cells (insulin), alpha-cells (glucagon), ...

Mouse Islet of Langerhans Isolation using a Combination of Purified Collagenase and Neutral Protease

The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines to the study of isolated rodent islets. Primary islets offer the distinct advantage that they more faithfully reflect the biology of intracellular signaling pathways and secretory responses. Whereas the method of islet isolation using tissue dissociating enzyme (TDE) preparations has been well established in many laboratories1-4, variations in the consistency of islet yield and quality from any given rodent strain limit the extent and feasibility of primary islet studies. These variations often occur as a result of the crude partially purified TDEs used in the islet isolation procedure; TDEs frequently exhibit lot-to-lot variations in activity and often require adjustments to the dose of enzyme used. A small number of reports have used purified TDEs for rodent cell isolations5, 6, but the practice is not widespread despite the routine use and advantages of purified TDEs for human islet isolations. In collaboration with VitaCyte, LLC (Indianapolis, IN), we developed a modified mouse islet isolation protocol based on that described by Gotoh7, 8, in which the TDEs are perfused directly into the pancreatic duct of mice, followed by crude tissue fractionation through a Histopaque gradient9, and isolation of purified islets. A significant difference in our protocol is the use of purified collagenase (CIzyme MA) and neutral protease (CIzyme BP) combination. The collagenase was characterized by the use of a6 fluorescence collagen degrading activity (CDA) assay that utilized fluorescently labeled soluble calf skin fibrils as substrate6. This substrate is more predictive of the kinetics of collagen degradation in the tissue matrix because it relies on native collagen as the substrate. The protease was characterized with a sensitive fluorescent kinetic assay10. Utilizing these improved assays along with more traditional biochemical analysis enable the TDE to be manufactured more consistently, leading to improved performance consistency between lots. The protocol described in here was optimized for maximal islet yield and optimal islet morphology using C57BL/6 mice. During the development of this protocol, several combinations of collagenase and neutral proteases were evaluated at different concentrations, and the final ratio of collagenase:neutral protease of 35:10 represents enzyme performance comparable to Sigma Type XI. Because significant variability in average islet yields from different strains of rats and mice have been reported, additional modifications of the TDE composition should be made to improve the yield and quality of islets recovered from different species and strains.

A detailed description of mouse islet isolation is described using the technique of in situ pancreatic ductal cannulation and perfusion of a combination of purified collagenase and neutral protease.

העכבר איילט בידוד נגרהנס באמצעות שילוב של collagenase המטוהר ופרוטאז הניטראלי

The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines ...

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one week. More than 20 x 106 acinar cells can be obtained from a single murine pancreas. This protocol offers the possibility to independently process as many as 10 pancreases in parallel. Because it preserves acinar architecture, this model is well suited for studying the physiology of the exocrine pancreas in vitro in contrast to cell lines established from pancreatic tumors, which display many genetic alterations resulting in partial or total loss of their acinar differentiation.

In this publication, we describe a rapid and convenient procedure for isolating and culturing primary pancreatic acinar cells from the murine pancreas. This method constitutes a valuable approach to study the physiology of fresh primary normal/untransformed exocrine pancreatic cells.

בידוד והתרבות של תאי לבלב הראשיים acinar עכבר

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

Near Infrared (NIr) Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium

Strategies for attenuating decline in balance function with increasing age are predominantly focused on physical therapies including balance tasks and exercise. However, these approaches do not address the underlying causes of balance decline. Using mice, the impact of near infrared light (NIr) on the metabolism of cells in the vestibular sensory epithelium was assessed. Data collected shows that this simple and safe intervention may protect these vulnerable cells from the deleterious effects of natural aging. mRNA was extracted from the isolated peripheral vestibular sensory epithelium (crista ampullaris and utricular macula) and subsequently transcribed into a cDNA library. This library was then probed for the expression of ubiquitous antioxidant (SOD-1). Antioxidant gene expression was then used to quantify cellular metabolism. Using transcranial delivery of NIr in young (4 weeks) and older (8 - 9 months) mice, and a brief treatment regime (90 sec/day for 5 days), this work suggests NIr alone may be sufficient to improve mitochondrial function in the vestibular sensory epithelium. Since there are currently no available, affordable, non-invasive methods of therapy to improve vestibular hair cell function, the application of external NIr radiation provides a potential strategy to counteract the impact of aging on cellular metabolism inthe vestibular sensory epithelium.

Near Infrared NIr Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium

Mitochondrial dysfunction is a hallmark of cellular senescence. This paper uses non-invasive near-infrared (NIr) treatment to improve mitochondrial function in the aging mouse vestibular sensory epithelium.

קרוב אינפרא אדום (ניר) אור מגביר את הביטוי של דה מרקר של פונקצית מיטוכונדריאלי בעכבר שיווי המשקל החושי האפיתל

Strategies for attenuating decline in balance function with increasing age are predominantly focused on physical therapies including balance tasks and ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

בידוד ותרבות של עכברים ראשיים Keratinocytes מ neonatal ומבוגרים עור עכבר

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other important biological functions. The islets primarily consist of five types of hormone-secreting cells: &#945; cells secrete glucagon, &#946; cells secrete insulin, &#948; cells secrete somatostatin, &#949; cells secrete ghrelin, and PP cells secrete pancreatic polypeptide. Sixty to 80% of the cells in the islets are &#946; cells, which are the most important cell population to study insulin secretion. Pancreatic islets are a crucial model system to study ex vivo insulin secretion. Acquiring high quality islets is of great importance for diabetes research. Most islet isolation procedures require technically difficult to access site of collagenase injection, harsh and complex digestion procedures, and multiple density gradient purification steps. This paper features a simple high yield mouse islet isolation method with detailed descriptions and realistic demonstrations, showing the following specific steps: 1) injection of collagenase P at the ampulla of Vater, a small area joining the pancreatic duct and the common bile duct, 2) enzymatic digestion and mechanical separation of the exocrine pancreas, and 3) a single gradient purification step. The advantages of this method are the injection of digestive enzyme using the more accessible ampulla of Vater, more complete digestion using combination of enzymatic and mechanical approaches, and a simpler single gradient purification step. This protocol produces approximately 250&#8212;350 islets per mouse; and islets are suitable for various ex vivo studies. Possible caveats of this procedure are potentially damaged islets due to enzymatic digestion and/or prolonged gradient incubation, all of which can be largely avoided by careful ad justification of incubation time.

This islet isolation protocol described a novel route of collagenase injection to digest the exocrine tissue and a simplified gradient procedure to purify the islets from mice. It involves enzymatic digestion, gradient separation/purification, and islet hand-picking. Successful isolation can yield 250&#8211;350 high quality and fully functional islets per mouse.

פרוטוקול פשוט יעילות גבוהה לבידוד איון הלבלב של עכברים

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other ...

A Method for Islet Transplantation to the Omentum in Mouse

Islet transplantation has been proposed to be a potential treatment for type 1 diabetes. Recent compelling evidence indicates that intravascular islet infusion is far from ideal and therefore, the omentum is re-emerging as a potentially valuable site for islet transplantation. This experiment requires the isolation of high quality islets and the implantation of the islets to the diabetic recipients. Transplantation to the omentum requires surgical steps that can be better demonstrated visually. Here, the detailed steps for this procedure are presented. Two methods of mixing the isolated islets with hydrogel before placing the mixture into the omental pouch of diabetic mice are described here. Different hydrogels are used for the different conditions. Blood glucose levels of diabetic mouse recipients of syngeneic islets in the omentum were monitored for up to 35 days. Some animals were sacrificed after 14 days to perform immuno-histochemical analysis. This pre-clinical transplantation approach can be used as preliminary data leading up to translation to clinical transplantation.

A method for the omental transplantation of islets in a mouse is introduced. The isolated islets are mixed with hydrogel and the mixture is placed into the omental pouch of a diabetic mouse. Then, the blood glucose is monitored, and immuno-histochemical analysis is performed.

שיטת השתלת איון כדי Omentum העכבר

Islet transplantation has been proposed to be a potential treatment for type 1 diabetes. Recent compelling evidence indicates that intravascular islet ...