עבודה זו נתמכה בחלקה על ידי המכונים הלאומיים לבריאות מענקים R21 AI113000 (WSM) ו R01 AI123392 (YF).

כל בעלי החיים המשמשים בניסויים שוכנו במתקן החיה במכון מחקר אלגני-זינגר תחת הפרוטוקול נבדק ואושר על ידי ועדת הטיפול בבעלי החיים המוסדיים השתמש המכון לחקר זינגר רשת בריאות אלגני / אלגני. 1. לעכל את התימוס עם Collagenase <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> בלוטות התימוס קציר. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> להרדים 3-5 שבועות בן עכברים C57BL6 / J בתוך הפחמן הדו-חמצני (CO 2) תא למסוק בלוטות התימוס. בפירוט, מניחים את העכברים בתא תחת אינדוקציה 2 CO 3-5 דקות ולאשר למוות על ידי אימות דום לב או נשימה. </li><li style=";text-align:right;direction:rtl"> הנח את הפגר על גבו להרטיב את הגוף עם אתנול 70%. לעשות חתך אופקי קטן עם מספרי לנתח חד, ישר על הבטן שלה דרך העור. משוך את העור משני הקצוות (ראש ורגליים), כך הוא גם הפוך לחלוטין. </li><li style=";text-align:right;direction:rtl"> ביצוע חתך אופקי עם המספריים לנתח רק תחתהצלע הנמוכה ביותר לחתוך דרך הסרעפת לצדדים. החזק את תהליך xiphoid עם מלקחיים ולחתוך באמצע הצלעות שתמשיך עד משני הצדדים. משוך כלפי מעלה את הצלעות / עצם החזה כך התימוס נראה בבירור. הערה: קורנית שוכנת ישירות על החלק העליון של הלב, מורכבת מ -2 אונות והוא בצבע לבן שקוף. </li><li style=";text-align:right;direction:rtl"> בעזרת מלקחיים מעוקלים, בעדינות לגרוף ולמשוך אונות התימוס ההנחה של רקמות חיבור העביר בתמיסת כביסה (ראה רשימה של חומרים). </li></ol></li><li style=";text-align:right;direction:rtl"> הכן פתרון העיכול על ידי ערבוב 9 מ&quot;ל RPMI-1640 (מכון ממוריאל פארק רוזוול בינוני-1640), 0.025 מ&quot;ג / collagenase מטוהרים מ&quot;ל, 10 HEPES מ&quot;מ [4- (2-hydroxyethyl) -1-piperazineethanesulfonic חומצה], ו -0.2 מ&quot;ג / מ&quot;ל DNase אני בתוך שפופרת 50 צנטריפוגות מ&quot;ל. שמור את הפתרון העיכול ב -37 מעלות צלזיוס המים באמבטיה עד השימוש. הערה: סכום פתרון העיכול המוכן מספיק thymi עכבר עד חמישה מבוגרים, אשר יכול להיות מודגרות יחד עגול קלקר אחד 5 מיליליטרצינור תחתון. </li><li style=";text-align:right;direction:rtl"> מעבירים את התימוס לצלחת בתרבית רקמה 60 מ&quot;מ המכיל 5-6 מ&quot;ל RPMI-1640. </li><li style=";text-align:right;direction:rtl"> לנתח את בלוטת התימוס עם 28 G אינסולין מזרק מחט לחתיכות קטנות (ריבוע 1 מ&quot;מ 3 על) כדי לאפשר לזרום החוצה לימפוציטים. </li><li style=";text-align:right;direction:rtl"> שוטפים את חתיכות הרתי עם RPMI-1640 כי הוא בצלחת פטרי עם פיפטה זכוכית פסטר. הטה את הצלחת ולתת שברי הרתי ליישב לתחתית. לאט לשאוב וזורקים supernatant RPMI-1640 באמצעות פיפטה מזכוכית. חזור על שלב שטיפה זה עם 5-6 מ&quot;ל RPMI-1640 עבור 4-5 פעמים עם פיפטה זכוכית עד הפתרון הוא פחות אטום. הערה: טפטפות זכוכית מומלץ בשלב זה, שכן שברי הרתי נוטים לדבוק פלסטיק, וכתוצאה מכך אובדן מוגזם של רקמות הרתי. עבור שאר ההליך, להשתמש טפטפות פלסטיק. </li><li style=";text-align:right;direction:rtl"> לאחר השטיפה הסופית, לבטל את supernatant כמתואר בשלב 1.5. הוסף פתרון העיכול 3 מ&quot;ל ולהעביר את חתיכות התימוס והפתרון לתוך 5 מ&quot;ל p מסביב לתחתיתצינור olystyrene. </li><li style=";text-align:right;direction:rtl"> מניחים את הצינור על הכתף צינור לדגור על 37 מעלות צלזיוס במשך 6 דקות במהירות סיבוב של 12 סל&quot;ד. </li><li style=";text-align:right;direction:rtl"> לאחר דגירה, לתת את שברי הרתי ליישב לתחתית של התחתית על ידי כוח הכבידה. אסוף את supernatant עם פיפטה פסטר ולהעביר לתוך צינור צנטריפוגה 50 מ&quot;ל עם 10 מ&quot;ל של תמיסת שטיפה. צנטריפוגה XG ב 300 במשך 5 דקות, לבטל את supernatant ו resuspend התאים בתמיסה לשטיפת 10 מ&quot;ל. שמור על הקרח. </li><li style=";text-align:right;direction:rtl"> חזור על שלבים 1.6-1.8 פעמיים על שברי הרתי ב -5 מ&quot;ל מסביב לתחתית צינור קלקר. לרכז את supernatant בצינור אותו 50 מ&quot;ל. הערה: צנטריפוגה אין צורך השני לשטוף השלישי. </li><li style=";text-align:right;direction:rtl"> לאחר העיכול הסופי, pipet למעלה ולמטה עם 2 מיליליטר פיפטה סרולוגיות פלסטיק לשבור כל שברים שנותרו בצינור והעברת צינור צנטריפוגות 50 המ&quot;ל. </li><li style=";text-align:right;direction:rtl"> צנטריפוגה שפופרת 50 מ&quot;ל ב 300 XG במשך 5 דקות, לשאוב supernatant, resuspend ב 10 מ&quot;ל של שטיפת buffer ולסנן דרך 100 מיקרומטר תא מסנן. שמור על הקרח. </li></ol> 2. העשרה של Tecs עם שיפוע צפיפות רציף <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכן פתרון שיפוע צפיפות 21% על ידי ערבוב 2.4 מ&quot;ל בינוני שיפוע צפיפות (60% w / v של iodixanol במים) פתרון הכביסה 9.6 מ&quot;ל. </li><li style=";text-align:right;direction:rtl"> צנטריפוגה התאים הרתי ניתק בצינור 50 מ&quot;ל אוסף משלב 1.11 ב XG 300 במשך 5 דקות ו resuspend במאגר כביסה 2.5 מ&quot;ל. </li><li style=";text-align:right;direction:rtl"> הוסף 20 מ&quot;ל בינוני RPMI-1640 על השעיית התא. </li><li style=";text-align:right;direction:rtl"> מלאו 12 מ&quot;ל של תמיסת שיפוע צפיפות 21% בתוך pipet סרולוגיות 10 מ&quot;ל עם פיפטור אלקטרוני. מניחים את קצה pipet סרולוגיות בתחתית הצינור צנטריפוגה 50 מ&quot;ל תאים המכילים, ולאפשר הפתרון שיפוע צפיפות לנקז אל החלק התחתון של הצינור באמצעות כוח הכבידה. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> בעדינות לגרש פתרון שיפוע צפיפות מ pipet לסיים שהניב שתי השכבות של צפיפויות שונות. </li></ol></li><li style=";text-align:right;direction:rtl"> צנטריפוגה ב 600 XG FOr 20 דקות ב RT ב הרוטור מתנדנד-דלי. הגדר את קצב ההאטה ברמה האיטית ביותר. </li><li style=";text-align:right;direction:rtl"> מעבירים את השכבה העליונה ואת ממשק לצינור חדש 50 מ&quot;ל. הערה: רוב Tecs נשמרים בממשק. לימפוציטים יהיה להאיץ לתחתית הצינור לאחר צנטריפוגה. אם תאים אלה ישמשו למטרות אחרות, יש לשטוף את הגלולה עם פתרון כביסה 20 מיליליטר במשך שלוש פעמים. Resuspend התאים 10 מ&quot;ל של חיץ הכביסה. </li><li style=";text-align:right;direction:rtl"> הוסף פתרון כביסה עד 40 מ&quot;ל אל הצינור בשלב 2.6 ו צנטריפוגות ב 400 XG במשך 6 דקות ב 4 ° C. כדי להסיר את supernatant, לשאוב עם pipet. </li><li style=";text-align:right;direction:rtl"> חזור על הכביסה פי 2 השאיפה כמתואר בשלב 2.7. </li><li style=";text-align:right;direction:rtl"> Resuspend ב 2 מ&quot;ל של תמיסת שטיפה לספור את התאים עם hemocytometer. </li></ol> 3. Tecs בידוד עם FACS <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> התאם התאים משלב 2.9 בריכוז של 1x10 6 תאים / 100 μl עם פתרון כביסה והעברתהתאים לצינור 5 מ&quot;ל מסביב לתחתית (פוליפרופילן או קלקר, כפי שהומלץ על ידי ההנחיות של סדרן זרימה). </li><li style=";text-align:right;direction:rtl"> מוסיפים 2 IgG הקולטן אנטי Fc μl לכל 1x10 6 תאים דגירה על 4 מעלות צלזיוס במשך 10 דקות. </li><li style=";text-align:right;direction:rtl"> תוספת CD45 אנטי (דילול 1: 150, 10 μl לדגימה) ואנטי-EpCAM (1: 100 דילול, 10 μl לדגימה) נוגדני דגירה על 4 מעלות צלזיוס במשך יותר מ -20 דקות. </li><li style=";text-align:right;direction:rtl"> שוטפים את התאים עם פתרון צנטריפוגות כביסה 2 מ&quot;ל ב 400 XG במשך 6 דקות ב 4 ° C. לשאוב supernatant עם טפטפת. </li><li style=";text-align:right;direction:rtl"> Resuspend התאים שנאגרו מלוחים פוספט 1x 1 מ&quot;ל (PBS). </li><li style=";text-align:right;direction:rtl"> סינון דרך מסננת 100 מיקרומטר. </li><li style=";text-align:right;direction:rtl"> החל את התאים למכשיר מיון. בחר את הלימפוציטים ואוכלוסיית TEC למעט התאים הקטנים ביותר על אור המפוזר בצד (SSC) / אור-מפוזר קדימה פנל (FSC) (תאים מתים), אז שער על האוכלוסייה שנבחרה CD45- EpCAM + למיון 13. </li></ol> 4. Generation של TEC / EAK אגרגטים הערה: יש לבצע הכנה מגיב וצעדים מעורבים תא ערבוב מתחת למכסה המנוע למינרית. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכן מתחמי מתאם PA / G על ידי ערבוב 4 μl של IgG אנטי-שלו-תג, 8 μl של IgG אנטי EpCAM ו -2.6 μl של הרשות / G בתוך שפופרת microcentrifuge סטרילי 1.5 מ&quot;ל, ו לדגור על RT במשך 5 דקות. </li><li style=";text-align:right;direction:rtl"> התאם את Tecs מסודרים משלב 3.7 ל 5x10 4 תאים / מ&quot;ל עם פתרון כביסה, ו pipet 100 μl כדי אחד טוב של אויר נמוך, 96-גם צלחת בתרבית רקמה מסביב לתחתית. להוסיף 14.6 מתחמי מתאם PA / G μl אל התאים. מניח את הצלחת על פלטפורמת נדנדה במשך 20 דקות ב 4 °. </li><li style=";text-align:right;direction:rtl"> שטפי פעם עם 200 פתרון הכביסה μl. צנטריפוגה את הצלחת ב 400 XG במשך 6 דקות. בטל supernatant עם micropipette. </li><li style=";text-align:right;direction:rtl"> שטפי פעם עם תמיסת סוכרוז 200 μl 10%. צנטריפוגה ב 400 XG במשך 6 דקות. בטל supernatant עם micropipette. </li><li style=";text-align:right;direction:rtl"> תאים Resuspend ב 30 μl 10% sתמיסת סוכרוז terile לכל טוב. </li><li style=";text-align:right;direction:rtl"> הוסף 10 μl של EAKIIH6 (7.5 מ&quot;ג / מ&quot;ל) ו דגירה במשך 5 דקות ב RT. </li><li style=";text-align:right;direction:rtl"> הוסף 20 μl של EAK16-II (10 מ&quot;ג / מ&quot;ל) לתערובת TEC. </li><li style=";text-align:right;direction:rtl"> הוספת 100 μl של מדיום מלא למערכת ליזום gelation. מניחים את הצלחת על שייקר פלטפורמה 15-20 דקות ב RT. </li><li style=";text-align:right;direction:rtl"> מניח את הצלחת בחממה על 37 מעלות צלזיוס, 5% CO 2. לאחר 2-4 שעות, להוסיף עוד 100 μl של בינוני מלא. </li><li style=";text-align:right;direction:rtl"> שנה בינונית בכל יום אחר עד השימוש. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לשאוב 100 μl של המדיום מפני השטח מבלי להפריע את הג&#39;ל באמצעות micropipette בעדינות להוסיף 100 μl של מדיום מראש חימם ידי הצבת קצה פיפטה אל הקיר של הבאר. </li></ol></li></ol> אפיון 5. של אגרגטים TEC / EAK <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> כדי לבחון את הפונקציה של אגרגטים TEC / EAK in vivo, לאסוף את TEC / הידרוג&#39;ל EAK לאחר O / N תרבות השתלת תחת הקפסולה כליות של athymic בעירוםעכבר, באמצעות שתואר לעיל הליך 11,14. </li><li style=";text-align:right;direction:rtl"> לפקח על התפתחות תאי T בפריפריה על ידי קצירת 50-100 דגימות דם μl לתוך צינור איסוף דם המכיל EDTA מן העכברים בעירום 4-6 שבועות לפרסם השתלה ואת כתם עם קוקטייל של CD4 אנטי, -CD8, -CD45 , ו -CD3 נוגדנים 12. </li><li style=";text-align:right;direction:rtl"> לנתח את האחוזים של לימפוציטים מסוג T ב לויקוציטים דם היקפיים עם cytometry זרימה (FCM), באמצעות תוכנת ניתוח תא בודד 12. </li></ol>

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Authors declare no conflict of interest.

בעוד Tecs הם האוכלוסייה השלטת של stroma הרתי ולשחק תפקידים חיוניים עבור המבנה והתפקוד של בלוטות התימוס, הם מהווים רק 0.1-0.5% של cellularity הרתי הכולל. הם גם תאי שברירי כמו באחוזים גבוהים של מוות של תאים הם נצפו לעתים בעקבות עיכול collagenase, כלומר, הטיפול לנתק Tecs מן מטריקס (ECM). הנד?...

התימוס הוא איבר הלימפה הראשי אחראי לייצור אוכלוסייה מגוונת של תאי הפתוגן-reactive, עצמי סובלני T כי הוא חיוני לתפקוד של מערכת החיסון הנרכשת. זהו איבר דינמי שבו thymocytes פיתוח, עלה ממח העצם כפי ובתאים הלימפוציטים, נודדים דרך מטריקס תלת מימדי דמוי ספוג (3-D) של stroma הרתי, לעבור השושלת-מפרט והבחנה, ובסופו של דבר להגר כפי בוגרת T-תאי. הצלחתו של תהליך מתוכנת היטב זה תלוי במידה רבה על הצטלבות בין thymocytes נודדות לתאי האפיתל מגורים הרתי (Tecs), האוכלוסייה השלטת של stroma הרתי החיוניים לקביעתם ולקיומם של שלמות במיקרו-סביבה של התימוס. בהתבסס על המיקום האנטומי שלהם פונקציה ייחודית, Tecs ניתן לחלק לשתי קבוצות משנה: הטקאנים בקליפה (cTECs) כי הם responsible לבחירה עצמי MHC (מורכבי histocompatibility גדולים) מוגבל מתאי T (סלקציה חיובית), ואת Tecs ב הלשד (mTECs) כי הם חיוניים עבור ביטול אוטוריאקטיבים תאי T (סלקציה שלילית) 1,2. גורמים רבים (למשל, הזדקנות, זיהום, קרינה, התרופה טיפולים) יכול לגרום נזק בלתי הפיך האפיתל הרתי, וכתוצאה מכך חסינות אדפטיבית נפגעת. למרות ניסיונות רבים, משחזר את תפקוד הרתי כבר מאתגר בשל הקושי לשחזר את המיקרו-סביבה הרתי. יש לציין, הרתי תלת ממדי (3-D) תצורה היא קריטית להישרדות והתפקוד של Tecs, ואילו Tecs תורבת בסביבת 2-D במהירות להקטין את הביטוי של גנים קריטיים עבור 3,4 thymopoiesis. EAK16-II (AEAEKAKAEAEAKAK) ו אנלוגי histidinylated C- מסוף שלה EAKIIH6 (AEAEKAKAEAEAKAKHHHHHH) הם משקל מולקולרי נמוך, oligopeptides amphiphilic כי הם מסיסים שתייה ללא יוניםr, אבל לעבור gelation ליצירת β-הסיבים כאשר הם נחשפים כוח יוניים גבוה מ -20 מ&quot;מ NaCl (ריכוז מלח רגיל בנוזל הגוף האנושי הוא 154 מ&quot;מ). נכס תגובה הסביבה זה גורם להם להיות אבני הבניין צדדי כדי ליצור מבנים 3D. The-התג שלו על EAKII-H6 מספק מנגנון עגינה, שבאמצעותו IgGs אנטי שלו / Fc מחייב חלבון רקומביננטי / G (αH6: PA / G) מתחמים יכולים לשמש מתאם לעגן תרופות חלבון ביומולקולות אחר ( למשל, נוגדנים) על מרוכבים הידרוג&#39;ל 5-8. אנחנו הוכחנו בעבר כי מולקולות ניאון שכותרתו IgG מעוגנות על הידרוג&#39;ל ניתן להיעזר באזור הזריקה עד 13 ימים 9. יתר על כן, כאשר αH6: מתאמי הרשות / G מעוגן עם IgGs אנטי CD4 נוספו EAK16-II / EAKIIH6 (EAK) הידרוג&#39;ל, תאי CD4 + T נתפסו במיוחד 10. באמצעות טכניקה דומה, אנחנו הוכחנו לאחרונה כי צבירת 3-D של ג Tecsולד יקודם הידרוג&#39;ל EAK עם מתחמי מתאם נושאת נוגדנים אנטי EpCAM TEC-ספציפי (αH6: PA / G: αEpCAM). כאשר מושתלים מתחת קפסולות הכליות של עכברים בעירום, אשכולות TEC מוטבעים הידרוג&#39;ל EAK יכולים לתמוך בפיתוח יעיל של תאי T התפקודיים in vivo 11,12. כאן אנו מדגימים את השיטה שלנו לטהר Tecs במהירות וביעילות עם תא המופעל הקרינה מיון (FACS) וליצור 3-D אגרגטים TEC עם מערכת הידרוג&#39;ל EAK.

<table border="0" cellpadding="0" cellspacing="0" width="722">
	<tbody>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">1. TEC Isolation</td>
			<td style="width:175px;"></td>
			<td style="width:111px;"></td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">70% Ethanol</td>
			<td>Decon Laboratories</td>
			<td>2701(1 Gallon)</td>
			<td style="width:223px;">Ethanol 200 Proof, deionized water</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">Dissecting scissors, straight</td>
			<td>Fine Science Tools, Inc.</td>
			<td>91460-11</td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">Graefe forceps, straight</td>
			<td>Fine Science Tools, Inc.</td>
			<td>11053-10</td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">Graefe forceps, curved</td>
			<td>Fine Science Tools, Inc.</td>
			<td>11052-10</td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:216px;">Washing solution</td>
			<td></td>
			<td></td>
			<td style="width:223px;">1X PBS, 0.1% BSA, 2mM EDTA</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:216px;">1x PBS (Phosphate buffered saline)</td>
			<td>Gibco</td>
			<td>10010-023</td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">BSA (Bovine serum albumin)</td>
			<td>Sigma</td>
			<td>A1470-100G</td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:47px;width:216px;">EDTA (ethylenediaminetetraacetic acid)</td>
			<td>Invitrogen</td>
			<td>15575-038</td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="66">
			<td height="66" style="height:67px;width:216px;">Digestion solution</td>
			<td></td>
			<td></td>
			<td style="width:223px;">9 mL RPMI-1640, 0.025 mg/mL Liberase TM Research grade, 10 mM HEPES, 0.2 mg/mL DNaseI</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">RPMI-1640</td>
			<td>Gibco</td>
			<td>11879-020</td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">Liberase TM Research Grade</td>
			<td>Roche</td>
			<td>05 401 127 001</td>
			<td style="width:223px;">referred as &#34;purified collagenase&#34;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">1M HEPES</td>
			<td>Lonza</td>
			<td>17-737E</td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">Dnase I</td>
			<td>Roche</td>
			<td>10 104 159 001</td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">50mL Centrifuge tube</td>
			<td>Corning</td>
			<td>430290</td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">60mm tissue culture dish</td>
			<td>Falcon</td>
			<td>353002</td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:216px;">1/2cc U-100 Insulin syringe 28G1/2</td>
			<td>Becton Dickinson</td>
			<td>329461</td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:216px;">5mL Polystyrene round-bottom tube</td>
			<td>Falcon</td>
			<td>352058</td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="62">
			<td height="62" style="height:63px;width:216px;">5ml glass pipet</td>
			<td>Fisher Healthcare</td>
			<td>13-678-27E</td>
			<td style="width:223px;">Use for rinsing the thymic fragments. Thymic fragments tend to stick to the wall with plastic pipets.</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">MACSmix tube rotator</td>
			<td>Miltenyi</td>
			<td>130-090-753</td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">100um Cell strainer</td>
			<td>Falcon</td>
			<td>352360</td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">Density gradient medium: OptiPrep</td>
			<td>Axis-Shield</td>
			<td></td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">Name</td>
			<td style="width:175px;">Company</td>
			<td style="width:111px;">Catalog Number</td>
			<td style="width:223px;">Comments</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">2. Cell sorting</td>
			<td></td>
			<td></td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;width:216px;">5mL Polypropylene round-bottom tube</td>
			<td>Falcon</td>
			<td>352063</td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">Anti-mouse CD16/CD32 (Fc Block)</td>
			<td>BD Biosciences</td>
			<td>553142</td>
			<td style="width:223px;">Use as undiluted, 2uL per sample</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">Anti-mouse CD45-PercpCy5.5</td>
			<td>eBioscience</td>
			<td>45-0451-80</td>
			<td style="width:223px;">Use at 1:150, 10uL per sample</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">Anti-mouse CD326 (EpCAM)-PE</td>
			<td>eBioscience</td>
			<td>12-5791-82</td>
			<td style="width:223px;">Use at 1:100, 10uL per sample</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">BD Influx</td>
			<td>BD Biosciences</td>
			<td></td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">Single cell analysis software</td>
			<td>FlowJo</td>
			<td></td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">Name</td>
			<td style="width:175px;">Company</td>
			<td style="width:111px;">Catalog Number</td>
			<td style="width:223px;">Comments</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">2. EAK gel assembly</td>
			<td></td>
			<td></td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">Anti-His-Tag</td>
			<td>AnaSpec</td>
			<td>29673</td>
			<td style="width:223px;">&#34;anti-His-Tag IgG&#34;</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:216px;">Purified anti-mouse CD326 (EpCAM)</td>
			<td>BioLegend</td>
			<td>118202</td>
			<td style="width:223px;">&#34;anti-EpCAM IgG&#34;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">Recombinant protein A/G</td>
			<td>Pierce Biotechnology</td>
			<td></td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="50">
			<td height="50" style="height:51px;width:216px;">1.5mL Safe-Lock Tubes, Biopur, Sterile</td>
			<td>Fisher Healthcare</td>
			<td>05-402-24B</td>
			<td style="width:223px;">referred as &#34;1.5mL microcentrifuge tube&#34;&#160;</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:216px;">96-well, Tissue culture plate, Round-bottom with low evaporation lid</td>
			<td>BD Falcon</td>
			<td>353917</td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Rocking platform: Nutator Mixer no.1105</td>
			<td>BD Clay Adams</td>
			<td></td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">10% sucrose</td>
			<td>Sigma</td>
			<td>S0389</td>
			<td style="width:223px;">Prepare with sterile distilled water</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">EAK16-II (AcNH-AEAEAKAKAEAEAKAK-CONH2)</td>
			<td>American Peptide Company&#160;</td>
			<td></td>
			<td style="width:223px;">custom synthesized, 10mg/mL</td>
		</tr>
		<tr height="58">
			<td height="58" style="height:59px;width:216px;">EAKIIH6 (AcNH-AEAEAKAKAEAEAKAKHHHHHH-CONH2)</td>
			<td>American Peptide Company&#160;</td>
			<td></td>
			<td style="width:223px;">custom synthesized, 7.5mg/mL</td>
		</tr>
		<tr height="76">
			<td height="76" style="height:76px;width:216px;">Complete medium</td>
			<td></td>
			<td></td>
			<td style="width:223px;">RPMI-1640, 10% FBS, 1% Pen/Strep, 1% L-glutamine, 1% NEAA, 5mM HEPES, 50uM 2-Mercaptoethanol</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">RPMI-1640&#160;</td>
			<td>Gibco</td>
			<td>11879-020</td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">FBS (Fetal Bovine Serum)</td>
			<td>Atlanta Biologicals</td>
			<td>S11150</td>
			<td style="width:223px;">Heat inactivated before use</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">Pen/Strep</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">L-glutamine 200mM (100x)</td>
			<td>Gibco</td>
			<td>25030-081</td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:216px;">NEAA (non-essential amino acid) 100x</td>
			<td>Gibco</td>
			<td>11140-050</td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">1M HEPES</td>
			<td>BioWhittaker</td>
			<td>17-737E</td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">2-Mercaptoethanol (100X)</td>
			<td>Millipore</td>
			<td>ES-007-E</td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Platform shaker: The Belly Dancer</td>
			<td>Stovall Life Sciences Inc.</td>
			<td>model: USBDbo</td>
			<td style="width:223px;"></td>
		</tr>
	</tbody>
</table>

promoting 3-d aggregation of facs purified thymic epithelial cells with eak 16-ii/eakiih6 self-assembling hydrogel

Thymus involution, associated with aging or pathological insults, results in diminished output of mature T-cells. Restoring the function of a failing thymus is crucial to maintain effective T cell-mediated acquired immune response against invading pathogens. However, thymus regeneration and revitalization proved to be challenging, largely due to the difficulties of reproducing the unique 3D microenvironment of the thymic stroma that is critical for the survival and function of thymic epithelial cells (TECs). We developed a novel hydrogel system to promote the formation of TEC aggregates, based on the self-assembling property of the amphiphilic EAK16-II oligopeptides and its histidinylated analogue EAKIIH6. TECs were enriched from isolated thymic cells with density-gradient, sorted with fluorescence-activated cell sorting (FACS), and labeled with anti-epithelial cell adhesion molecule (EpCAM) antibodies that were anchored, together with anti-His IgGs, on the protein A/G adaptor complexes. Formation of cell aggregates was promoted by incubating TECs with EAKIIH6 and EAK16-II oligopeptides, and then by increasing the ionic concentration of the medium to initiate gelation. TEC aggregates embedded in EAK hydrogel can effectively promote the development of functional T cells in vivo when transplanted into the athymic nude mice.

כדי לבחון את האפקטיביות של השימוש בפרוטוקול ההפרדה שיפוע צפיפות להעשיר את תאי סטרומה CD45-, תאים שנקטפו משני הממשק ואת כדורי הלימפוציטים זירז הוכתמו אנטי CD45 ונוגדנים אנטי EpCAM. שני 1 אנטי Ulex Europaeus agglutinin (UEA1) ונוגדנים נגד MHC Class II נכללו גם קוקטייל מכתים להמ?...

Watch this Scientific Journal Video about Promoting 3-D Aggregation of FACS Purified Thymic Epithelial Cells with EAK 16-II/EAKIIH6 Self-assembling Hydrogel at JoVE.com

Promoting 3-D Aggregation of FACS Purified Thymic Epithelial Cells with EAK 16-II/EAKIIH6 Self-assembling Hydrogel

This video demonstrates a protocol to enrich thymic epithelial cells (TECs) with density gradient for FACS isolation. It also shows the use of EAK16-II/EAKIIH6 peptides to promote the TEC aggregate formation. The microenvironments of EAK16-II/EAKIIH6 hydrogel provide the 3-D configuration necessary to maintain the survival and function of the TECs.

קידום Aggregation 3-D של תאי אפיתל הרתי המטוהר FACS עם EAK 16-II / EAKIIH6 עצמית להרכבת הידרוג&#39;ל

Thymus involution, associated with aging or pathological insults, results in diminished output of mature T-cells. Restoring the function of a failing ...

promoting-3-d-aggregation-facs-purified-thymic-epithelial-cells-with

Research

JoVE Journal

Immunology and Infection

6.5K Views.  Allegheny Health Network. This video demonstrates a protocol to enrich thymic epithelial cells (TECs) with density gradient for FACS isolation. It also shows the use of EAK16-II/EAKIIH6 peptides to promote the TEC aggregate formation. The microenvironments of EAK16-II/EAKIIH6 hydrogel provide the 3-D configuration necessary to maintain the survival and function of the TECs.

Video: Promoting 3-D Aggregation of FACS Purified Thymic Epithelial Cells with EAK 16-II/EAKIIH6 Self-assembling Hydrogel

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS)

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known phenotype.  Other bulk methods of purification include panning, complement depletion and magnetic bead separation.  However, FACS has several advantages over other available methods.  FACS is the preferred method when very high purity of the desired population is required, when the target cell population expresses a very low level of the identifying marker or when cell populations require separation based on differential marker density.  In addition, FACS is the only available purification technique to isolate cells based on internal staining or intracellular protein expression, such as a genetically modified fluorescent protein marker.  FACS allows the purification of individual cells based on size, granularity and fluorescence.  In order to purify cells of interest, they are first stained with fluorescently-tagged monoclonal antibodies (mAb), which recognize specific surface markers on the desired cell population (1).  Negative selection of unstained cells is also possible.  FACS purification requires a flow cytometer with sorting capacity and the appropriate software.  For FACS, cells in suspension are passed as a stream in droplets with each containing a single cell in front of a laser.  The fluorescence detection system detects cells of interest based on predetermined fluorescent parameters of the cells.  The instrument applies a charge to the droplet containing a cell of interest and an electrostatic deflection system facilitates collection of the charged droplets into appropriate collection tubes (2).  The success of staining and thereby sorting depends largely on the selection of the identifying markers and the choice of mAb.  Sorting parameters can be adjusted depending on the requirement of purity and yield.  Although FACS requires specialized equipment and personnel training, it is the method of choice for isolation of highly purified cell populations.

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting FACS

For many scientific studies requiring a biological and chemical analysis of cell populations the cells must be in a high state of purity. Fluorescence activated cell sorting (FACS) is a superior method in which to obtain pure cell populations.

טיהור אוכלוסייה תאים ספציפיים על ידי מיון פלואורסצנטי תא פעיל (FACS)

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known ...

Induction and Assessment of Class Switch Recombination in Purified Murine B Cells

Humoral immunity is the branch of the immune system maintained by B cells and mediated through the secretion of antibodies. Upon B cell activation, the immunoglobulin locus undergoes a series of genetic modifications to alter the binding capacity and effector function of secreted antibodies. This process is highlighted by a genomic recombination event known as class switch recombination (CSR) in which the default IgM antibody isotype is substituted for one of IgG, IgA, or IgE. Each isotype possesses distinct effector functions thereby making CSR crucial to the maintenance of immunity. Diversification of the immunoglobulin locus is mediated by the enzyme activation-induced cytidine deaminase (AID). A schematic video describing this process in detail is available online (http://video.med.utoronto.ca/videoprojects/immunology/aam.html). AID's activity and the CSR pathway are commonly studied in the assessment of B cell function and humoral immunity in mice. The protocol outlined in this report presents a method of B cell isolation from murine spleens and subsequent stimulation with bacterial lipopolysaccharide (LPS) to induce class switching to IgG3 (for other antibody isotypes see Table 1). In addition, the fluorescent cell staining dye Carboxyfluorescein succinimidyl ester (CFSE) is used to monitor cell division of stimulated cells, a process crucial to isotype switching 1, 2. The regulation of AID and the mechanism by which CSR occurs are still unclear and thus in vitro class switch assays provide a reliable method for testing these processes in various mouse models. These assays have been previously used in the context of gene deficiency using knockout mice 3. Furthermore, in vitro switching of B cells can be preceded by viral transduction to modulate gene expression by RNA knockdown or transgene expression 4-6. The data from these types of experiments have impacted our understanding of AID activity, resolution of the CSR reaction, and antibody-mediated immunity in the mouse.

Following antigen exposure, subpopulations of activated B cells undergo a process known as class switch recombination (CSR) to produce antibody isotypes with distinct effector functions. The protocol outlined in this report explains how CSR can be induced and analyzed in vitro for the purposes of studying B cell function.

אינדוקציה והערכה של רקומבינציה החלף מחלקה ב מטוהרים תאים B Murine

Humoral immunity is the branch of the immune system maintained by B cells and mediated through the secretion of antibodies. Upon B cell activation, the ...

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host cells1. These adhesins rely on interactions with host cell surface receptors or soluble proteins acting as a bridge between bacteria and host. Adhesion is a critical first step prior to invasion and/or secretion of toxins, thus it is a key event to be studied in bacterial pathogenesis. Furthermore, adhered bacteria often induce exquisitely fine-tuned cellular responses, the studies of which have given birth to the field of 'cellular microbiology'2. Robust assays for bacterial adhesion on host cells and their invasion therefore play key roles in bacterial pathogenesis studies and have long been used in many pioneer laboratories3,4. These assays are now practiced by most laboratories working on bacterial pathogenesis.
Here, we describe a standard adherence assay illustrating the contribution of a specific adhesin. We use the Escherichia coli strain 27875, a human pathogenic strain expressing the autotransporter Adhesin Involved in Diffuse Adherence (AIDA). As a control, we use a mutant strain lacking the aidA gene, 2787&Delta;aidA (F. Berthiaume and M. Mourez, unpublished), and a commercial laboratory strain of E. coli, C600 (New England Biolabs). The bacteria are left to adhere to the cells from the commonly used HEp-2 human epithelial cell line. This assay has been less extensively described before6.

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

This protocol is a simple bacterial adhesion assay consisting in counting the numbers of bacterial colony forming units that are adhered onto cultured cells. The assay is robust, independent of the adhesin studied, and numerous variations are used in most laboratories working on bacterial pathogenesis.

ב Assay חוץ גופית של הידבקות חיידקים על תאים אפיתל היונקים

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host ...

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed to them and occasionally to pathogens. The gut-associated lymphoid tissue (GALT) play a key role for induction of the mucosal immune response to these microbes1, 2. To initiate the mucosal immune response, the mucosal antigens must be transported from the gut lumen across the epithelial barrier into organized lymphoid follicles such as Peyer's patches. This antigen transcytosis is mediated by specialized epithelial M cells3, 4. M cells are atypical epithelial cells that actively phagocytose macromolecules and microbes. Unlike dendritic cells (DCs) and macrophages, which target antigens to lysosomes for degradation, M cells mainly transcytose the internalized antigens. This vigorous macromolecular transcytosis through M cells delivers antigen to the underlying organized lymphoid follicles and is believed to be essential for initiating antigen-specific mucosal immune responses. However, the molecular mechanisms promoting this antigen uptake by M cells are largely unknown. We have previously reported that glycoprotein 2 (Gp2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane 5. Here, we present a method for the application of a mouse Peyer's patch intestinal loop assay to evaluate bacterial uptake by M cells. This method is an improved version of the mouse intestinal loop assay previously described 6, 7. The improved points are as follows: 1. Isoflurane was used as an anesthetic agent. 2. Approximately 1 cm ligated intestinal loop including Peyer's patch was set up. 3. Bacteria taken up by M cells were fluorescently labeled by fluorescence labeling reagent or by overexpressing fluorescent protein such as green fluorescent protein (GFP). 4. M cells in the follicle-associated epithelium covering Peyer's patch were detected by whole-mount immunostainig with anti Gp2 antibody. 5. Fluorescent bacterial transcytosis by M cells were observed by confocal microscopic analysis. The mouse Peyer's patch intestinal loop assay could supply the answer what kind of commensal or pathogenic bacteria transcytosed by M cells, and may lead us to understand the molecular mechanism of how to stimulate mucosal immune system through M cells.

Application of a Mouse Ligated Peyer&#8217;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

M cells in a specialized follicle-associated epithelium covering Peyer&#x2019;s patches play an important role for the mucosal immunosurveillance in gut-associated lymphoid tissue. Here we described the evaluation method for bacterial transcytosis by M cells in vivo. This method provides a method to understand M-cell function in the immune system.

יישום של עכבר Ligated מעיים תיקון של Peyer Assay Loop להערכת ספיגת חיידקים על ידי תאים M

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

Examination of Thymic Positive and Negative Selection by Flow Cytometry

A healthy immune system requires that T cells respond to foreign antigens while remaining tolerant to self-antigens. Random rearrangement of the T cell receptor (TCR) &alpha; and &beta; loci generates a T cell repertoire with vast diversity in antigen specificity, both to self and foreign. Selection of the repertoire during development in the thymus is critical for generating safe and useful T cells. Defects in thymic selection contribute to the development of autoimmune and immunodeficiency disorders1-4. 
T cell progenitors enter the thymus as double negative (DN) thymocytes that do not express CD4 or CD8 co-receptors. Expression of the &alpha;&beta;TCR and both co-receptors occurs at the double positive (DP) stage. Interaction of the &alpha;&beta;TCR with self-peptide-MHC (pMHC) presented by thymic cells determines the fate of the DP thymocyte. High affinity interactions lead to negative selection and elimination of self-reactive thymocytes. Low affinity interactions result in positive selection and development of CD4 or CD8 single positive (SP) T cells capable of recognizing foreign antigens presented by self-MHC5.
Positive selection can be studied in mice with a polyclonal (wildtype) TCR repertoire by observing the generation of mature T cells. However, they are not ideal for the study of negative selection, which involves deletion of small antigen-specific populations. Many model systems have been used to study negative selection but vary in their ability to recapitulate physiological events6. For example, in vitro stimulation of thymocytes lacks the thymic environment that is intimately involved in selection, while administration of exogenous antigen can lead to non-specific deletion of thymocytes7-9. Currently, the best tools for studying in vivo negative selection are mice that express a transgenic TCR specific for endogenous self-antigen. However, many classical TCR transgenic models are characterized by premature expression of the transgenic TCR&alpha; chain at the DN stage, resulting in premature negative selection. Our lab has developed the HYcd4 model, in which the transgenic HY TCR&alpha; is conditionally expressed at the DP stage, allowing negative selection to occur during the DP to SP transition as occurs in wildtype mice10.
Here, we describe a flow cytometry-based protocol to examine thymic positive and negative selection in the HYcd4 mouse model. While negative selection in HYcd4 mice is highly physiological, these methods can also be applied to other TCR transgenic models. We will also present general strategies for analyzing positive selection in a polyclonal repertoire applicable to any genetically manipulated mice.

We present a flow cytometry-based method to examine T cell development in vivo using genetically manipulated mice on a wildtype or T cell receptor transgenic background.

בחינה של בחירה חיובית ושלילית הרתי ידי זרימת cytometry

A healthy immune system requires that T cells respond to foreign  antigens while remaining tolerant to self-antigens. Random rearrangement of the  T cell ...

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem cells are responsible for ensuring tissue homeostasis and repair throughout life, it is reasonable to hypothesize that the stem cell population is critical for preserving genomic integrity of tissues. Therefore, significant interest has arisen in assessing the impact of endogenous and environmental factors on genomic integrity in stem cells and their progeny, aiming to understand the etiology of stem-cell based diseases.
LacI transgenic mice carry a recoverable &#955;&#160;phage vector encoding the LacI reporter system, in which the LacI gene serves as the mutation reporter. The result of a mutated LacI gene is the production of &#946;-galactosidase that cleaves a chromogenic substrate, turning it blue. The LacI reporter system is carried in all cells, including stem/progenitor cells and can easily be recovered and used to subsequently infect E. coli. After incubating infected E. coli on agarose that contains the correct substrate, plaques can be scored; blue plaques indicate a mutant LacI gene, while clear plaques harbor wild-type. The frequency of blue (among clear) plaques indicates the mutant frequency in the original cell population the DNA was extracted from. Sequencing the mutant LacI gene will show the location of the mutations in the gene and the type of mutation.
The LacI transgenic mouse model is well-established as an in vivo mutagenesis assay. Moreover, the mice and the reagents for the assay are commercially available. Here we describe in detail how this model can be adapted to measure the frequency of spontaneously occurring DNA mutants in stem cell-enriched Lin-IL7R-Sca-1+cKit++(LSK) cells and other subpopulations of the hematopoietic system.

Here we describe an in vivo mutagenesis assay for small numbers of purified hematopoietic cells using the LacI transgenic mouse model.&#160;The LacI gene can be isolated to determine the frequency, location, and type of DNA mutants&#160;spontaneously arisen or after exposure to genotoxins.

זיהוי מוטציות DNA בתאי גזע / אב המטוהר Hematopoietic

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

בידוד של תאי מיאלואידית דנדריטים ותאי האפיתל מקורנית האדם

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

בידוד, זיהוי, וטיהור של תאי Murine הרתי אפיתל

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Isolation and Transplantation of Different Aged Murine Thymic Grafts.

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and selection of T cells specific for self and foreign antigens. The approach entails implantation of thymic grafts derived from various aged mice into immunodeficient scid recipients. Over a relatively short period of time the recipients are fully reconstituted with T cells derived from the implanted thymus graft. Only thymocytes seeding the thymus at the time of isolation undergo selection and develop into mature T cells. As such, changes in the nature and specificity of the engrafted T cells as a function of age-dependent thymic events can be assessed. Although technical expertise is required for successful thymic transplantation, this method provides a unique strategy to study in vivo a wide range of pathologies that are due to or a result of aberrant thymic function and/or homeostasis.

This report provides a detailed description of transplanting murine thymi from different aged donor mice under the kidney capsule of immunodeficient mouse recipients. The goal of this approach is to model T cell development and thymic selection events in vivo.

בידוד והשתלה של שתלי הרתי Murine בני שונים.

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and ...

Preparation and Applications of Organotypic Thymic Slice Cultures

Thymic selection proceeds in a unique and highly organized thymic microenvironment resulting in the generation of a functional, self-tolerant T cell repertoire. In vitro models to study T lineage commitment and development have provided valuable insights into this process. However, these systems lack the complete three-dimensional thymic milieu necessary for T cell development and, therefore, are incomplete approximations of in vivo thymic selection. Some of the challenges related to modeling T cell development can be overcome by using in situ models that provide an intact thymic microenvironment that fully supports thymic selection of developing T cells. Thymic slice organotypic cultures complement existing in situ techniques. Thymic slices preserve the integrity of the thymic cortical and medullary regions and provide a platform to study development of overlaid thymocytes of a defined developmental stage or of endogenous T cells within a mature thymic microenvironment. Given the ability to generate ~20 slices per mouse, thymic slices present a unique advantage in terms of scalability for high throughput experiments. Further, the relative ease in generating thymic slices and potential to overlay different thymic subsets or other cell populations from diverse genetic backgrounds enhances the versatility of this method. Here we describe a protocol for the preparation of thymic slices, isolation and overlay of thymocytes, and dissociation of thymic slices for flow cytometric analysis. This system can also be adapted to study non-conventional T cell development as well as visualize thymocyte migration, thymocyte-stromal cell interactions, and TCR signals associated with thymic selection by two-photon microscopy.

We describe the preparation of thymic slices that, in combination with flow cytometry, can be used to model positive and negative selection of developing T cells. Thymic slices can also be adapted for the in situ analysis of thymocyte migration, localization, and signaling via immunofluorescence and two-photon microscopy.

הכנה ויישומים של תרבויות Organotypic הרתי Slice

Thymic selection proceeds in a unique and highly organized thymic microenvironment resulting in the generation of a functional, self-tolerant T cell ...