Diese Arbeit wurde zum Teil durch die National Institutes of Health gewährt R21 AI113000 (WSM) und R01 AI123392 (YF) unterstützt.

Alle Tiere in den Experimenten verwendet wurden in der Tierhaltung bei Allegheny-Singer Research Institute im Rahmen des Protokolls überprüft und genehmigt von der Institutional Animal Care und Use Committee des Allegheny Health Network / Allegheny Singer Research Institute untergebracht. 1. Verdauen des Thymus mit Kollagenase <ol><li> Ernte Thymusdrüse. <ol><li> Euthanize 3-5 Wochen alte C57BL6 / J - Mäusen in einem Kohlendioxid (CO 2) Kammer Thymusdrüse zu ernten. Im Einzelnen stellen die Mäuse in der Kammer unter CO 2 Induktion für 3-5 min und Tod bestätigen durch Herz- oder Atemstillstand zu überprüfen. </li><li> Legen Sie die Karkasse auf den Rücken und nass, den Körper mit 70% Ethanol. Machen Sie einen kleinen horizontalen Schnitt mit einem scharfen, geraden Präparierscheren auf seinem Bauch durch die Haut. Ziehen Sie die Haut an beiden Enden (Kopf und Beine), so dass es von innen nach außen gedreht wird. </li><li> Machen Sie einen horizontalen Schnitt mit den Dissektionsscheren knappdie niedrigste Rippe und seitlich durch die Membran geschnitten. Halten Sie die Xiphoidbasis mit einer Pinzette und schneiden Sie die Mitte der Rippen auf beiden Seiten ausgehend nach oben. Ziehen Sie die Rippen / Brustbein nach oben, so dass der Thymus deutlich sichtbar ist. Hinweis: Thymus direkt auf der Oberseite des Herzens liegt, besteht aus zwei Lappen und einer weißen durchscheinenden Farbe. </li><li> Unter Verwendung einer gebogenen Pinzette vorsichtig schöpfen und die Thymus Lappen weg von dem Bindegewebe und den Transfer in Waschlösung ziehen (siehe Materialliste). </li></ol></li><li> Vorbereitung Aufschlußlösung durch Mischen von 9 ml RPMI-1640 (Rpmi-1640), 0,025 mg / ml gereinigter Kollagenase, 10 mM HEPES [4- (2-Hydroxyethyl) -1-piperazinethansulfonsäure] und 0,2 mg / ml DNase I in einem 50 ml Zentrifugenröhrchen. Halten Sie die Aufschlusslösung in 37 ° C warmen Wasserbad bis zur Verwendung. Anmerkung: Die Menge der Aufschlußlösung ausreichend vorbereitet ist für bis zu fünf erwachsenen Maus Thymi, die zusammen Polystyrol rund- in einem 5 ml inkubiert werden können,Unterrohr. </li><li> Übertragen Sie die Thymusdrüse zu einer 60 mm-Gewebekulturschale mit 5-6 ml RPMI-1640. </li><li> Präparieren Sie die Thymusdrüse mit 28 G Insulinspritze Nadel in kleine Stücke (ca. 1 mm 3 Quadrat) die Lymphozyten zu lassen abfließen. </li><li> Spülen Sie die Thymus-Stücke mit RPMI-1640, das mit Glas Pasteur Pipette in der Petrischale ist. Kippen Sie die Schüssel und lassen Sie die Thymus-Fragmente am Boden absetzen. Langsam ansaugen und das RPMI-1640-Überstandes mit einer Glaspipette zu verwerfen. Wiederholen Sie diesen Spülschritt mit 5-6 ml RPMI-1640 für 4-5 mal mit Glaspipette, bis die Lösung weniger undurchsichtig ist. Hinweis: Glaspipetten werden bei diesem Schritt zu empfehlen, da Thymus-Fragmente zu Kunststoff Verkleben neigen, was zu einem übermäßigen Verlust von Thymus-Gewebe. Für den Rest des Verfahrens, verwenden Sie Plastikpipetten. </li><li> Nach dem letzten Spülgang, den Überstand verwerfen, wie in Schritt 1.5 beschrieben. 3 ml Aufschlusslösung und übertragen Sie die Thymusdrüse Stücke und die Lösung in einen 5 ml Rundboden-polystyrene Rohr. </li><li> Das Röhrchen wird auf einem Rohr Rotator und inkubieren bei 37 ° C für 6 min bei einer Rotationsgeschwindigkeit von 12 Umdrehungen pro Minute. </li><li> Nach der Inkubation ließ die Thymus-Fragmente auf den Boden des Rohres durch die Schwerkraft absetzen. Sammeln Sie den Überstand mit Pasteur-Pipette und Überführung in eine 50 ml-Zentrifugenröhrchen mit 10 ml Waschlösung. Zentrifuge bei 300 g für 5 min den Überstand verwerfen und Resuspendieren der Zellen in 10 ml Waschlösung. Halten Sie sich auf dem Eis. </li><li> Wiederholen Sie die Schritte 1,6-1,8 zweimal auf den Thymus-Fragmente in den 5 ml Rundboden-Polystyrolröhrchen. Pool den Überstand in der gleichen 50-ml-Tube. Anmerkung: Die Zentrifugation nicht notwendig, für die zweite und die dritte Waschung. </li><li> Nach der endgültigen Verdauung, pipettieren nach oben und unten mit 2 ml Kunststoff serologischen Pipette auf die Zentrifugenröhrchen 50 ml alle verbleibenden Fragmente in der Röhre und den Transfer zu brechen. </li><li> Zentrifuge das 50 ml-Röhrchen bei 300 xg für 5 min, Überstand absaugen, Resuspendieren in 10 ml Wasch buffer und filtriert durch 100 &amp; mgr; m Zellsieb. Halten Sie sich auf dem Eis. </li></ol> 2. Anreicherung von TECs mit unstetigen Dichtegradienten <ol><li> Vorbereitung 21% Dichtegradient-Lösung von 2,4 ml Dichtegradientenmedium Mischen (60% w / v von Iodixanol in Wasser) und 9,6 ml Waschlösung. </li><li> Zentrifugieren Sie die dissoziierten Thymus-Zellen in der 50 ml Sammelröhrchen aus Schritt 1.11 bei 300 g für 5 min und Resuspendieren in 2,5 ml Waschpuffer. </li><li> 20 ml RPMI-1640-Medium zu der Zellsuspension. </li><li> Füllen 12 ml 21% Dichtegradient-Lösung in einem 10 ml serologische Pipette mit einer elektronischen Pipettiervorrichtung. Die Spitze der serologischen Pipette am Boden der 50-ml-Zentrifugenröhrchen, das Zellen und ermöglichen der Dichtegradient-Lösung auf den Boden des Rohres durch Schwerkraft zu entleeren. <ol><li> Sanft auszustoßen, die zwei Schichten unterschiedlicher Dichte zu beenden die Dichtegradientenlösung aus der Pipette zu erzeugen. </li></ol></li><li> Zentrifuge bei 600 xg for 20 min bei RT in einem Schwenkbecherrotor. Stellen Sie die Verzögerungsrate am langsamsten Niveau. </li><li> Übertragen Sie die obere Schicht und die Schnittstelle zu einem neuen 50-ml-Tube. Hinweis: Die meisten der TECs sind in der Schnittstelle beibehalten. Lymphozyten werden dem Boden des Röhrchens nach der Zentrifugation auszufällen. Wenn diese Zellen für andere Zwecke verwendet werden sollen, spült das Pellet mit 20 ml Waschlösung dreimal. Resuspendieren der Zellen in 10 ml Waschpuffer. </li><li> Hinzufügen Waschlösung in das Röhrchen zu 40 ml bis in Schritt 2.6 und Zentrifuge bei 400 × g für 6 Minuten bei 4 ° C. Zu dem Überstand absaugen mit einer Pipette entfernen. </li><li> Wiederholen Sie das Waschen und die Streben 2 weitere Male, wie in Schritt 2.7 beschrieben. </li><li> Resuspension in 2 ml Waschlösung und der Zellen mit einem Hämozytometer zählen. </li></ol> 3. Isolierkanne TECs mit FACS <ol><li> Einstellen Zellen aus Schritt 2.9 in der Konzentration von 1x10 6 Zellen / 100 &amp; mgr; l mit Waschlösung und Transferdie Zellen in einem 5 ml-Rundbodenrohr (Polypropylen oder Polystyrol, wie durch die Anweisungen des Flusssortierer empfohlen). </li><li> Fügen Sie 2 &amp; mgr; l anti-Fc - Rezeptor - IgG pro 1x10 6 Zellen und Inkubieren bei 4 ° C für 10 min. </li><li> Hinzufügen anti-CD45 (1: 150 Verdünnung, 10 ul pro Probe) und anti-EpCAM (1: 100-Verdünnung, 10 ul pro Probe) Antikörper und Inkubieren bei 4 ° C für mehr als 20 min. </li><li> Waschen der Zellen mit 2 ml Waschlösung und Zentrifugation bei 400 × g für 6 Minuten bei 4 ° C. Saugen Sie den Überstand mit einer Pipette. </li><li> Resuspendieren der Zellen in 1 ml 1x phosphatgepufferter Kochsalzlösung (PBS). </li><li> Man filtriert durch 100 &amp; mgr; m Sieb. </li><li> Anwenden, um die Zellen auf die Sortieranlage. Wählen Sie die Lymphozyten und TEC Bevölkerung mit Ausnahme der kleinsten Zellen auf Seitenstreulicht (SSC) / Vorwärtsstreulicht (FSC) Platte (tote Zellen), dann Tor auf der ausgewählten Population für CD45- EpCAM + 13 Sortierung. </li></ol> 4. Generation von TEC / EAK Aggregates Hinweis: Führen Sie Reagenzzubereitung und Schritten Zelle unter der laminaren Haube zu mischen. <ol><li> Bereiten pA / G Adaptor-Komplexe durch Mischen von 4 &amp; mgr; l anti-His-Tag-IgG, 8 &amp; mgr; l anti-EpCAM IgG und 2,6 ul pA / G in ein steriles 1,5 ml Mikrozentrifugenröhrchen, und Inkubation für 5 min bei RT. </li><li> Passen Sie die sortiert TECs aus Schritt 3,7 bis 5x10 4 Zellen / ml mit Waschlösung, und pipettieren 100 &amp; mgr; l zu einer Vertiefung einer niedrigen Verdampfungs, 96-Well - Rundboden - Gewebekulturplatte. Hinzufügen 14,6 ul pA / G Adaptor-Komplexe zu den Zellen. Legen Sie die Platte auf einer Schwingplattform für 20 min bei 4 ° C. </li><li> Waschen Sie einmal mit 200 &amp; mgr; l Waschlösung. Zentrifugieren Sie die Platte bei 400 × g für 6 min. Entsorgen Sie den Überstand mit einer Mikropipette. </li><li> Wasche einmal mit 200 ul 10% igen Saccharoselösung. Zentrifuge bei 400 × g für 6 min. Entsorgen Sie den Überstand mit einer Mikropipette. </li><li> Resuspendiere Zellen in 30 &amp; mgr; l 10% sterile Saccharoselösung pro Vertiefung. </li><li> Fügen Sie 10 &amp; mgr; l EAKIIH6 (7,5 mg / ml) und Inkubation für 5 min bei RT. </li><li> Geben Sie 20 &amp; mgr; l EAK16-II (10 mg / ml) zu der Mischung TEC. </li><li> Füge 100 &amp; mgr; l vollständigem Medium in das System Gelbildung einzuleiten. Legen Sie die Platte auf einem Plattformschüttler für 15-20 min bei RT. </li><li> Die Platte wird in den Inkubator bei 37 ° C und 5% CO 2. Nach 2-4 Stunden, fügen Sie weitere 100 &amp; mgr; l Vollmedium. </li><li> Ändern Medium jeden zweiten Tag bis zum Gebrauch. <ol><li> Absaugen 100 ul des Mediums von der Oberfläche, ohne das Gel zu stören mit einer Mikropipette und fügen Sie vorsichtig 100 ul vorgewärmte Medium durch die Pipettenspitze an der Wand des Bohrlochs platzieren. </li></ol></li></ol> 5. Charakterisierung der TEC / EAK Aggregates <ol><li> Um die Funktion der TEC / EAK - Aggregate in vivo zu untersuchen, sammeln die TEC / EAK - Hydrogel nach O / N - Kultur und Transplantation unter die Nierenkapsel einer athymischen NacktMaus unter Verwendung von zuvor Prozedur 11,14 beschrieben. </li><li> Überwachen Sie die Entwicklung von T-Zellen in der Peripherie durch Ernten 50-100 ul Blutproben in EDTA-enthaltenden Blutentnahmeröhrchen aus dem nackten Mäusen 4-6 Wochen nach der Transplantation und Flecken mit einem Cocktail aus anti-CD4, -CD8, -CD45 und -CD3 Antikörper 12. </li><li> Analysieren die Prozentsätze von T-Lymphozyten in den peripheren Blutleukozyten mit Durchflusszytometrie (FCM), unter Verwendung von Einzelzellanalyse - Software 12. </li></ol>

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Authors declare no conflict of interest.

Während TECs die vorherrschende Bevölkerung der Thymus-Stroma und spielen eine entscheidende Rolle für die Struktur und Funktion der Thymusdrüse sind, repräsentieren sie nur etwa 0,1-0,5% des gesamten Thymus Zellularität. Sie sind auch zerbrechlich Zellen als hohe Anteile an Zelltod werden gelegentlich folgenden Kollagenaseverdau beobachtet, dh die Behandlung TECs aus der extrazellulären Matrix (ECM) zu dissoziieren. Ihre Seltenheit (~ 200.000 pro Maus Thymus) und Zerbrechlichkeit machte es eine anspruch...

Der Thymus ist die primäre lymphoiden Organ für die Erzeugung einer vielfältigen Population von pathogen-reaktives, selbst tolerant T-Zellen, die für die Funktion des erworbenen Immunsystems wesentlich ist. Es ist ein dynamisches Organ, in dem die Entwicklung Thymozyten aus dem Knochenmark als Lymphozyten-Vorläuferzellen immigrierte, wandern durch die schwammartige dreidimensionale (3-D) Matrix des Thymus-Stroma, durchlaufen abstammungs Spezifizierung und Differenzierung, und schließlich auswandern als reifen T-Zellen. Der Erfolg dieses gut programmiert Verfahren hängt weitgehend von der Kreuzkopplung zwischen den wandernden Thymozyten und den Wohn Thymus-Epithelzellen (TECs), die vorherrschende Bevölkerung des Thymus-Stroma, die für die Einrichtung und Aufrechterhaltung der Integrität des Thymus Mikroumgebung wesentlich sind. Auf der Grundlage ihrer anatomischen Lage und einzigartige Funktion kann TECs in zwei Untergruppen unterteilt werden: die TECs in der Hirnrinde (CTEC), die Verant sindwortlich Selbst MHC (Major Histocompatibility Complex) beschränkt T-Zellen (positive Auswahl) und die TECs in der Medulla (mTEZ) , die zur Eliminierung autoreaktiver T-Zellen wesentlich sind (negative Selektion) 1,2 für die Auswahl. Viele Faktoren (zB Alterung, Infektion, Bestrahlung, medikamentöse Behandlungen) können irreversible Schäden an der Thymus - Epithel führen, in kompromittierten adaptive Immunität führt. Trotz zahlreicher Versuche hat die Thymusfunktion Wiederherstellung herausfordernd aufgrund der Schwierigkeit, den Thymus-Mikroumgebung zu reproduzieren. Bemerkenswerterweise ist thymic dreidimensionale (3-D) -Konfiguration entscheidend für das Überleben und die Funktion von TECs, während TECs kultiviert in einem 2-D - Umgebung schnell die Expression von Genen kritisch für Thymopoese 3,4 verringern. EAK16-II (AEAEKAKAEAEAKAK) und dessen C-terminale histidinylated analogen EAKIIH6 (AEAEKAKAEAEAKAKHHHHHH) sind niedermolekulare, amphiphile Oligopeptide, die in deionisiertem gegen Ende der löslichenr, sondern gehen Gelieren β-Fibrillen zu bilden, wenn sie Ionenstärke von mehr als 20 mM NaCl (normale Salzkonzentration in der menschlichen Körperflüssigkeit beträgt 154 mM) ausgesetzt. Diese Umwelt reagierende Eigenschaft macht sie vielseitige Bausteine ​​3D-Strukturen zu bilden. Das His-Tag auf EAKII-H6 ein Andockmechanismus, durch welche die anti-His IgGs / Fc-bindenden rekombinantes Protein A / G (αH6: pA / G) -Komplexen als Adapter dienen können Proteinwirkstoffen und anderen Biomolekülen zu verankern ( zB Antikörper) auf dem Hydrogelverbundstoff 5-8. Wir haben zuvor gezeigt , daß 9 bis zu 13 Tage an der Injektionsstelle fluoreszenzmarkierte IgG - Moleküle auf dem Hydrogel verankert können beibehalten werden. Wenn ferner die αH6: pA / G Adapter verankert mit anti-CD4 IgGs an das Hydrogel EAK16-II / EAKIIH6 (EAK) hinzugefügt wurden, wurden CD4 + T - Zellen spezifisch 10 eingefangen. Unter Verwendung ähnlicher Technik haben wir vor kurzem gezeigt, dass 3-D-Aggregation von TECs c(: PA / G: αEpCAM αH6) Ould mit Adapter Komplexe tragen TEC-spezifische anti-EpCAM-Antikörper in EAK Hydrogel gefördert werden. Wenn unterhalb der Nierenkapseln von nackten Mäusen transplantiert, können die TEC - Cluster in EAK Hydrogel eingebettet effektiv die Entwicklung von funktionellen T - Zellen in vivo 11,12 unterstützen. Hier veranschaulichen wir unser Verfahren, um schnell und effektiv reinigen TECs mit fluoreszenzaktivierter Zellsortierung (FACS) und Erzeugen 3-D TEC Aggregate mit dem EAK Hydrogelsystem.

<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.

Um die Wirksamkeit der Verwendung der Dichtegradiententrennung Protokoll untersuchen die CD45- Stromazellen zu bereichern, geerntete Zellen, sowohl von der Oberfläche und den ausgefällten Lymphozyten Pellets wurden mit anti-CD45 und anti-EpCAM-Antikörper. Sowohl anti-Ulex Europaeus Agglutinin 1 (UEA1) und Anti-MHC-Klasse II-Antikörper wurden auch in der Färbungs Cocktail aufgenommen, um die cTEC und mTEC Untergruppen identifizieren. Wie in Abbildung 1 gezeigt, konnt...

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.

Die Förderung der 3-D-Aggregation von FACS Purified Thymusepithelzellen mit EAK 16-II / EAKIIH6 Selbstaufbau Hydrogels

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 Aufrufe.  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.

Serie: 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.

Reinigung von spezifischen Zellpopulation durch 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 ...

Forschung

Immunologie und Infektion

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.

Induktion und Beurteilung der Class Schalter Rekombination in Purified Murine B-Zellen

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.

In-vitro-Assay der bakteriellen Adhäsion auf Mammalian Epithelzellen

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.

Die Anwendung eines Maus Ligierte Peyer-Patch-Darmschlinge Assay zu bakterielle Aufnahme von M-Zellen auswerten

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.

Prüfung Thymic positive und negative Selektion mittels Durchflusszytometrie

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.

Identifizierung von DNA-Mutationen in hämatopoetischen Stammzellen Gereinigtes / Vorläuferzellen

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.

Isolierung von myeloider dendritischer Zellen und Epithelzellen Menschliche 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 ...

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.

Isolierung, Identifizierung und Reinigung von Murine Thymusepithelzellen

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.

Isolation und Transplantation von 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 ...

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.

Herstellung und Bedeutung von Organotypischen Thymic Scheibe Kulturen

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