Name: isolation and activation of murine lymphocytes
Uploaded: 2016-10-30T14:00:00.000+00:00
Duration: 8 min 8 s
Description: Watch this Scientific Journal Video about Isolation and Activation of Murine Lymphocytes at JoVE.com

Die Studie wird durch das Ministerium für Bildung, Singapur (ACRF Tier1-RG40 / 13 und Tier2-MOE2013-T2-2-038) unterstützt. Das Manuskript wurde von Amy Sullivan aus Obrizus Kommunikation bearbeitet.

Alle Mäuse gezüchtet und unter spezifischen pathogenfreien Bedingungen und alle Maus-Protokolle werden in Übereinstimmung mit den Richtlinien der Institutional Animal Care und Use Committee geführt gehalten. 1. Herstellung von Puffer und Reagenzien <ol><li> Bereiten vollständige Roswell Park Memorial Institute (RPMI) Medium (10% hitzeinaktiviertem fötalem Rinderserum (FBS), 2 mM L-Glutamin, Penicillin (100 IU / ml) / Streptomycin (100 ug / ml), 55 uM 2-Mercaptoethanol ). </li><li> Bereiten Sie 20x Balanced Salt Solution (BSS) Lager 1 und Lager 2, getrennt. <ol><li> Bereiten Sie 20x BSS Lager 1 (111 mM Dextrose, 8,8 mM Kaliumphosphat, 26,7 mM Dinatriumhydrogenphosphat in 1 l sterilem Wasser) und 40 ml 0,5% Phenolrot bis 20x BSS Lager 1 vor Endvolumeneinstellung. Sterilfilter unter Verwendung von 0,2-um-Filter, bevor bei 4 ° C lagern. </li><li> Bereiten Sie 20x BSS Lager 2 (25,8 mM Calciumchloriddihydrat, 107 mM Kaliumchlorid, 2,73 M Natrium chloride, 19,6 mM Magnesiumchlorid-Hexahydrat, 16,6 mM Magnesiumsulfat in 1 l sterilem Wasser). Sterilfilter unter Verwendung von 0,2-um-Filter, bevor bei 4 ° C lagern. </li><li> 1x BSS für den experimentellen Einsatz, verdünnen 50 ml BSS Lager 1 und 50 ml BSS Lager 2, getrennt, in 400 ml sterilem Wasser je vorzubereiten. Kombinieren Sie beide verdünnten Lösungen, passen auf pH 7 und 20 ml FBS (2%). Top bis zu 1 L mit sterilem Wasser und Sterilfilter mit 0,2 um Filter. HINWEIS: BSS Stammlösungen separat hergestellt werden sollte, weil die Mischung von konzentrierter BSS Aktien direkt in Ausfällen führen kann. </li></ol></li><li> Rote Blutkörperchen (RBC) Lysepuffer <ol><li> RBC Lysispuffer, mischen 9 Teile Lager Ammoniumchlorid (155 mM Ammoniumchlorid in sterilem Wasser) zu 1 Teil Lager Tris-Base (130 mM Tris (hydroxymethyl) aminomethan in sterilem Wasser, pH-Wert 7,65) vor dem Gebrauch zu bereiten. HINWEIS: Shop sterilen Stammlösungen von Ammoniumchlorid und Tris-Base bei 4 ° C. Bereiten Sie die Lyse bUffer frisch effiziente Lyse von roten Blutkörperchen zu gewährleisten. </li></ol></li></ol> 2. Erzeugung von Lymphozytensuspension aus Milz oder Lymphknoten Hinweis: Es ist wichtig, dass alle Reagenzien und Geräte für das Experiment vor Maus Euthanasie und zur Erzeugung Einzelzellsuspensionen von Lymphozyten so schnell wie möglich zu halten hohe Zellviabilitäten erforderlich vorzubereiten. <ol><li> Euthanize experimentellen Maus durch Genickbruch oder CO 2 Ersticken. HINWEIS: Von diesem Schritt an gelten alle experimentellen Verfahren unter aseptischen Bedingungen durchgeführt werden sollte. </li><li> Tauchen Sie die gesamte Maus in 70% Ethanol, bevor irgendwelche Schnitte zu machen. Entfernen Sie die Milz und Lymphknoten aseptisch 8, und legen Sie sie in einem separaten 15 - ml - Röhrchen , die 5 ml eiskaltem RPMI / FBS (RPMI mit 2% FBS) oder BSS / FBS (aus Schritt 1.2.3). HINWEIS: Da BSS effiziente RBC-Lyse verhindert, verwenden RPMI für die Herstellung der Milz- Zellsuspension und wechseln Sie zu BSS nacr RBC-Lyse. </li><li> Um eine einzelne Zellsuspension aus der Milz oder Lymphknoten, legen Sie das Organ (e) zwischen zwei Stücken von sterilen 100 &amp; mgr; m Zellsieb mesh in einer Petrischale mit 2 ml eiskaltem RPMI / FBS oder BSS / FBS erzeugen. Mit dem Kolben einer 1-ml-Spritze, zerdrücken die Orgel (n), bis er in sehr feine Teile zerrissen wurde. </li><li> Übertragen Sie die Zellsuspension in ein 15-ml-Röhrchen und waschen Sie die Zelle Sieb mit eiskaltem RPMI / FBS oder BSS / FBS Netz. Sammeln Sie die restlichen Zellsuspension, fügen Sie ihn auf den gleichen 15-ml-Tube und Spin-down bei 453 × g für 5 Minuten bei 4 ° C. Entfernen Sie den Überstand. HINWEIS: Die pelletierten Zellen erneut zu suspendieren, indem das Rohr mit den Fingern Ausklopfen vor RBC-Lyse Puffer oder Medium in den nachfolgenden Schritten hinzugefügt wird. </li><li> Vorbereitung Raumtemperatur (RT) RBC-Lyse-Puffer während der Zentrifugation der Zellsuspension (siehe Schritt 1.3.). Nachdem die Zellen pelletiert und Entfernen des Überstands, resuspendieren der Zellen mit 1 ml Puffer RBC - Lyse je 10 8 Zellen. Incubate die Lysereaktion bei RT für 3-4 min. </li><li> Stopp RBC Lyse mit 14 ml eiskaltem BSS / FBS und Spin-down bei 453 × g für 5 Minuten bei 4 ° C. </li></ol> 3. Reinigung von B und T-Zellen <ol><li> Reinigung von B-Zellen <ol><li> Zählen Sie die Zellen mit einer Zählkammer. Re-suspendieren bis zu 10 8 Milzzellen in 300 &amp; mgr; l BSS / FBS und 50 &amp; mgr; l anti-CD43 9,10 magnetischen Mikrokügelchen hinzuzufügen. Zum Entfernen von toten Zellen, fügen Sie 30 ul Annexin V magnetischen Kügelchen. Inkubieren der Zellsuspension in einem 4 ° C Kühlschrank für 30 min. </li></ol></li><li> Reinigung von T-Zellen <ol><li> Zählen Sie die Zellen mit einer Zählkammer. Re-suspendieren bis zu 10 8 Zellen in nicht-T - Zell - Depletion - Antikörper - Cocktail (biotinylierter Antikörper gegen CD19, B220, Gr-1, TCR-γδ, CD49b, CD11c, CD11b, Ter119 und CD4 oder CD8 abhängig von der Zielzellpopulation gereinigt werden), 1: 200 verdünnt in 200 &amp; mgr; l BSS / FBS 4,5. Inkubieren der Zellsuspension in ein4 ° C Kühlschrank für 15 min. </li><li> Nach der Inkubation, 10 ml BSS / FBS die Zellen und Spin-down bei 453 × g für 5 Minuten bei 4 ° C zu waschen. Entfernen Sie den Überstand und resuspendieren die Zellen in 165 &amp; mgr; l BSS / FBS mit 30 &amp; mgr; l Streptavidin-Mikrokügelchen und 15 &amp; mgr; l Annexin V magnetischen Kügelchen. Inkubieren der Zellsuspension in einem 4 ° C Kühlschrank für 30 min. HINWEIS: Um sicherzustellen, dass auch mit den magnetischen Mikrokügelchen Markierung, die Zellen mit Microbeads für 15 min inkubieren, dann die Zellsuspension vorsichtig mischen, indem die 15-ml-Röhrchen klopfen und weitere 15 min bei Schritt 3.1.1 inkubiert. oder 3.2.2. </li></ol></li><li> Herstellung von Trennsäule für Zellreinigung <ol><li> Bereiten Sie eine nicht verwendete Trennsäule während Mikrobead Markierung der Zellen (Schritt 3.1.1. Oder 3.2.2.). Vorwärmen BSS (ohne FBS) auf RT und verwenden 2 ml die Säule aseptisch zu waschen und ins Gleichgewicht. Nach Äquilibrierung mit BSS sollte die gewaschene Säule nicht austrocknen gelassen werden. HINWEIS: Wir verwenden die LS cPALTE anstelle der Spalte empfohlen LD aufgrund seiner Wiederverwendbarkeit (siehe Schritt 3.4.). </li><li> Nach der Markierung mit den magnetischen Kügelchen, fügen Sie 14 ml BSS / FBS die Zellen und Spin-down bei 453 × g für 5 Minuten bei 4 ° C zu waschen. Entfernen Sie den Überstand und erneut die Zellen in 1-3 ml RT BSS. </li><li> Befestigen eines sterilen 21 G Nadel an der Spitze der Säule, die Durchflussrate während des Prozesses der Reinigung zu reduzieren. Laden die Zellsuspension auf die äquilibrierte Säule und Sammeln des Durchflusses durch die gereinigten Zielzellen enthält. HINWEIS: Vermeiden Sie Blasen in die Spalte während des Ladens einzuführen. </li><li> Die Säule wird einmal mit 1 ml BSS / FBS und sammle die Strömung durch die gereinigten Zielzellen enthält. Neuladen der Säule mit dem Fluss durch wieder. Sammeln Sie die Strömung durch nach dem zweiten Laden in der gleichen 15-ml-Tube. </li><li> Die Säule wird 3-mal mit 1 ml BSS / FBS und sammeln die Strömung durch die gereinigten Zielzellen enthält. Danach werden 5 ml BSS / FBS zu the-Säule und mit einem Kolben, spülen Sie die magnetisch markierten Zellen aus der Säule in ein neues 15-ml-Tube. </li><li> Überprüfen Sie die Reinheit der durch Strömungs gesammelten Zellen Zytometrie unter Verwendung von Antikörpern , die Antigene binden , 4,5 gereinigtes B oder T - Zellen an die Oberfläche. </li></ol></li><li> Die Wiederverwendung von der Trennsäule HINWEIS: Die LS Spalte bis 4 Mal wiederverwendet werden kann, ohne Reinigungswirkungsgrad zu beeinträchtigen. <ol><li> Die Säule wird 3-mal mit 5 ml phosphatgepufferter Salzlösung (PBS) und 3-mal mit 5 ml destilliertem Wasser von oben den Kolben verwendet wird. </li><li> Die Säule wird mit 5 ml 70% Ethanol und trocknet die Säule ausgiebig einen Lufthahn mit dem Aufbau von Rost in der Säule zu verhindern. </li><li> Um eine gebrauchte LS-Säule für eine separate Reinigung Experiment vorzubereiten, waschen Sie die Säule von unten nach oben mit 5 ml 70% Ethanol mit dem Spritzenadapter. Als nächstes waschen Sie die Spalte von unten einmal von der Spitze der Säule mit 5 ml PBS zweimal mit 5 ml sterilem PBS, gefolgt. In 2ml RT BSS die Säule und dann fahren Sie mit dem Laden der Säule mit markierten Zellen zu äquilibrieren. </li></ol></li></ol> 4. CFSE Labeling und Stimulation HINWEIS: Gereinigte Zellen können auf eine Vielzahl von in vitro und in vivo funktionellen Assays unterzogen werden. Hier verwenden wir gereinigten T - Zellen , die T - Zell - Stimulation Fähigkeit von APCs 5 zu bestimmen. <ol><li> Vorwärmen Markierungslösung (0,1% FBS in PBS) auf 37 ° C vor der CFSE Belastung. HINWEIS: einen geringen Prozentsatz an FBS in PBS Verwendung reduziert Zelltod während CFSE Laden und minimiert Zellverlust während der Zentrifugation. Doch zu viel FBS kann mit CFSE Laden stören. </li><li> Bei 2 x 10 7 Zellen / ml in vorgewärmten Markierungslösung in einem Rohr 15 ml Waschen gereinigt Zellen zweimal mit Markierungslösung, dann wieder suspendiert. </li><li> Bereiten Sie 10 &amp; mgr; M CFSE-Lösung (1: 500 Verdünnung von 5 mM CFSE Stammlösung) in vorgewärmten Markierungslösung. CFSE Lösung solltewerden jeweils frisch zu erreichen optimale Kennzeichnung vorbereitet. </li><li> Um Zellen mit CFSE laden, fügen 1 Teil Zellsuspension zu 1 Teil 10 &amp; mgr; M CFSE-Lösung in einem 15-ml-Röhrchen und Inkubation im Dunkeln für 10 min bei 37 ° C. Eine Endkonzentration von 5 &amp; mgr; M CFSE verwendet ml zu etikettieren 1 x 10 7 Zellen /. </li><li> Drehen Sie das Röhrchen alle 2 Minuten eine homogene Mischung von Zellen während der CFSE Belastung zu gewährleisten. </li><li> Um die Reaktion zu stoppen, mehrere Bände eiskaltem komplettem RPMI Medium hinzufügen und Spin-down bei 453 × g für 5 Minuten bei 4 ° C. Nach dem erfolgreichen CFSE Laden wird das Zellpellet gelblich erscheinen. </li><li> Wash CFSE Zellen ein weiteres Mal mit eiskaltem kompletten RPMI - Medium und Spin - down bei 453 × g für 5 Minuten bei 4 ° C geladen , bevor sie für in - vitro - Kultivierung oder in vivo Stimulation verwendet wird . </li></ol> 5. In - vitro - Stimulation <ol><li> Bereiten Sie eine 2x-Stammlösung von Reizen (2x Stimuli Stammlösung) unmittelbar vor der Anwendung, so dass 100 &amp; mgr; l 2x Stimuli Stammlösung kann in einer 96-Well-Platte zu 100 ul Zellen zu einem Endvolumen von 200 &amp; mgr; l pro Vertiefung zugegeben werden. </li><li> Wenn eine Platte mit Stimuli (IgM oder CD40 für B-Zellen oder CD3 und CD28 für T-Zellen), aufgetragen erforderlich ist, verdünnt die Stimuli in PBS und pre-coat der Kulturplatte bei 4 ° C über Nacht. Alternativ kann der Kulturplatte bei 37 ° C für 1 Stunde am Tag des Versuchs überzogen werden. Waschen Sie die beschichtete Platte zweimal mit PBS (Nicht in die Platte zu jeder Zeit zu trocknen). </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> Für B - Zellen </td><td></td></tr><tr><td> Stimuli </td><td> Die Endkonzentration </td></tr><tr><td> F (ab &#39;) 2 Ziegen-anti-Maus-IgM </td><td> 0,6-2,4 &amp; mgr; g / ml </td></tr><tr><td> Anti-Maus-CD40 mAb </td><td> 0,5-2 &amp; mgr; g / ml </td></tr><tr><td> RekombinanteMaus-IL-4 </td><td> 25 U / ml </td></tr><tr><td> Lipopolysaccharide </td><td rowspan="2"> 0,1-10 &amp; mgr; g / ml </td></tr><tr><td> (LPS) aus E. coli Serotyp 055: B5 </td></tr><tr><td></td><td></td></tr><tr><td> Für T - Zellen </td><td></td></tr><tr><td> Stimuli </td><td> Die Endkonzentration </td></tr><tr><td> Anti-CD3 (Platte beschichtet) </td><td> 2-10 &amp; mgr; g / ml </td></tr><tr><td> (50 &amp; mgr; l / Vertiefung für die Beschichtung) </td><td></td></tr><tr><td> Anti-CD28 (Platte beschichtet) </td><td> 2 &amp; mgr; g / ml </td></tr><tr><td> Rekombinantes IL-2 </td><td> 40 U / ml </td></tr><tr><td> PDBu (Phorbol ester) </td><td> 5-50 ng / ml </td></tr><tr><td> A23187 (Calciumionophor) </td><td> 250 ng / ml </td></tr></tbody></table> Tabelle 1: Die Konzentrationen von Stimuli Lymphozyten in in - vitro - Kultur zu stimulieren.<ol start="3"><li> Für CFSE-markierten B - Zellen, resuspendieren bis 3 x 10 6 Zellen / ml in komplettem RPMI - Medium und der Kultur in dreifacher Ausfertigung mit 3 x 10 5 Zellen / Vertiefung in 96-well - Flachbodenplatten für 72 Stunden. </li><li> Für CFSE-markierten T - Zellen, resuspendieren zu 0,5-3 x 10 6 Zellen / ml in komplettem RPMI - Medium und der Kultur in dreifacher Ausführung mit 0,5-3 x 10 5 Zellen / Well in 96-Well - Rundbodenplatten für 48 oder 72 Std . </li></ol> 6. In - vivo - Stimulation <ol><li> Für in vivo Stimulation, adoptiv Übertragungs 4 x 10 6 CFSE-markierten T - Zellen pro Maus (intravenös (iv) in 200 &amp; mgr; l PBS) in jedes MHC-angepaßten Empfängermaus. HINWEIS: In diesem Protokoll CFSE-markierten T-Zellen adoptiv übertragen werden können Schwanzvene oder retroorbitale Injektions da diese Zellen verwendet werden Hause Organe wie die Milz und Lymphknoten lymphoiden. </li><li> Fordern Sie die Empfängermäuse 1 Tag später mit dem Antigen. HINWEIS:In diesem Beispiel verwenden wir Ovalbumin (OVA-Protein, 50 &amp; mgr; g / Maus) als Antigen weil OVA-spezifischen T-Zell-Rezeptor (TCR) -transgene T-Zellen adoptiv in Empfängermäuse transferiert. Bereiten Sie OVA Protein in sterilem PBS und injizieren 100 ul OVA Protein / PBS oder PBS - Kontrolle wird über eine subkutane Injektion (si), in jede Empfängermaus 5. </li><li> Ernte und Einzelzellsuspensionen aus lymphatischen Organen (Lymphknoten und Milz) erzeugen von Empfängermäuse 3 Tage nach der Immunisierung mit OVA-Protein oder PBS. Separate Lymphknoten in proximal-Lymphknoten (PLN), die Achsel umfasst, brachialis und oberflächlicher Lymphknoten) und distale Lymphknoten (dLN), die mesenterischen umfasst, popliteal, inguinal, Lenden- und caudal Lymphknoten. Stain Zellen die entsprechenden FACS unter Verwendung von Antikörpern für T-Zellproliferation zu überprüfen. HINWEIS: In diesem CFSE Zelle Tracking-Experiment, CFSE-markierten T-Zellen von PBS-injizierten Kontrollmäusen eine Fluoreszenzbasis für Nicht-di etablierenViding Zellen. Zellteilungen von proliferierenden, Antigen-stimulierten, CFSE-markierten Zellen werden durch Messen Fluoreszenzpeaks 12,13 visualisiert. Mit der PBS - Kontrolle, die Anzahl der Zellteilungen von proliferierenden, CFSE-markierten T - Zellen können 12,13 bestimmt werden. </li><li> Analyse der CFSE-markierten Zellproliferationsdaten durch die Anzahl der Zellteilungen zu vergleichen oder Spitzen zwischen den Proben (1 und 2). HINWEIS: Zum Beispiel CFSE-markierte, OVA-spezifischen T - Zellen adoptiv Empfängermäuse übertragen in das OVA - Antigen empfangen wird aktiv proliferieren im Vergleich zu den PBS-injizierten Kontrollmäusen 5,12. Darüber hinaus ist es bemerkenswert , darauf hinweisen , dass es viele Wege gibt CFSE Zellproliferationsdaten zu analysieren, wie 14 von Hawkins und Kollegen gezeigt. </li></ol>

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The authors have nothing to disclose.

In diesem Protokoll zeigen wir ein Verfahren für die Lymphozyten aus lymphoiden Organen zu reinigen. Zellreinigung magnetischen Kügelchen Sortieranlage verwendet, ist eine schnelle und einfache Methode, die lebensfähige, hochgereinigten Zielzellen ergibt. Kritische Schritte im Rahmen des Protokolls Die Lebensfähigkeit der Zellen und Zellausbeute Die Aufrechterhaltung Lebensfähigkeit von hämatopoet...

Mature lymphocytes generally exist in the resting state if there is no pre-existing infection or inflammation in the individual. Therefore, it is important to retain the na&#239;ve status of lymphocytes during the isolation process before performing in vitro or in vivo functional assays. The key to ensuring consistent and reproducible results is to limit any unnecessary manipulation of the cells. 
Magnetic cell sorting utilizes antibodies and microbeads to label cells so as to enrich the cell population of interest. With this approach, there are two purification strategies: positive enrichment and negative depletion. Positive enrichment enriches the cell population of interest using an antibody that binds to the target cells. Negative depletion, on the other hand, depletes non-target cells, leaving the cell population of interest. In our lab, we prefer negative depletion to positive enrichment because the binding of antibodies to the target cells could potentially alter cell features and behavior. In fact, many established cell surface markers suitable for the isolation of a particular cell population are also functional receptors. 
Magnetic cell sorting not only yields highly pure populations of viable target cells, it is also less time-consuming and avoids the cellular stress induced by high-pressure flow used in fluorescence-activated cell sorting (FACS). By labeling the unwanted cell populations and depleting them using a magnetic separation column, we are able to perform rapid cell isolation without compromising the viability of the target cell population. In this protocol, we demonstrate the use of negative depletion strategies to purify na&#239;ve B cells or T cells.

<table><tbody><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>RPMI 1640 (without L-Glutamine)</td><td>Gibco</td><td>31870025</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td></td><td></td><td>Heat inactivated&amp;nbsp;</td></tr><tr><td>L-glutamine</td><td>Gibco</td><td>25030024</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Gibco</td><td>15140114</td><td></td></tr><tr><td>2-mercaptoethanol</td><td>Gibco</td><td>21985023</td><td></td></tr><tr><td>Anti-CD43 magnetic microbeads</td><td>Miltenyi Biotec</td><td>130-049-801</td><td>Mix well prior use</td></tr><tr><td>Streptavidin microbeads</td><td>Miltenyi Biotec</td><td>130-048-101</td><td>Mix well prior use</td></tr><tr><td>Anti-Annexin V magnetic beads</td><td>Miltenyi Biotec</td><td>130-090-201</td><td>Mix well prior use</td></tr><tr><td>MACS LD&amp;nbsp;</td><td>Miltenyi Biotec</td><td>130-042-901</td><td></td></tr><tr><td>96-well U-bottom sterile culture plate</td><td>Greiner Bio-one</td><td>650180</td><td></td></tr><tr><td>96-well F-bottom sterile culture plate</td><td>Greiner Bio-one</td><td>655180</td><td></td></tr><tr><td>100 &amp;mu;m cell strainer mesh</td><td></td><td></td><td>To sterilize using UV radiation prior use</td></tr><tr><td>0.2 &amp;mu;m&amp;nbsp; sterile disposable filter units</td><td>Nalgene</td><td>567-0020</td><td>Can be substituted with any sterile filter device</td></tr><tr><td>CellTrace Violet</td><td>Invitrogen</td><td>C34557</td><td>CTV for short; alternative to CFSE</td></tr><tr><td>CellTrace Yellow</td><td>Invitrogen</td><td>C34567</td><td>CTY for short; alternative to CFSE</td></tr><tr><td>CellTrace Far Red</td><td>Invitrogen</td><td>C34564</td><td>CTFR for short; alternative to CFSE</td></tr><tr><td>Cell Proliferation Dye eFluor 670</td><td>eBioscience</td><td>65-0840</td><td>CPD670 for short; alternative to CFSE</td></tr><tr><td>PKH26</td><td>Sigma Aldrich</td><td>PKH26GL</td><td>PKH26, alternative to CFSE</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Chemicals&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Dextrose</td><td>Sigma Aldrich</td><td>G7021</td><td></td></tr><tr><td>Potassium phosphate monobasic</td><td>Sigma Aldrich</td><td>P5655</td><td></td></tr><tr><td>Sodium phosphate dibasic</td><td>Sigma Aldrich</td><td>S5136</td><td></td></tr><tr><td>Phenol Red</td><td>Sigma Aldrich</td><td>P0290</td><td></td></tr><tr><td>Calcium chloride dihydrate</td><td>Sigma Aldrich</td><td>C7902</td><td></td></tr><tr><td>Potassium chloride</td><td>Sigma Aldrich</td><td>P5405</td><td></td></tr><tr><td>Sodium chloride</td><td>Merck Millipore</td><td>S7653</td><td>Can use from other sources</td></tr><tr><td>Magnesium chloride hexahydrate</td><td>Sigma Aldrich</td><td>M2393</td><td></td></tr><tr><td>Magnesium sulfate</td><td>Sigma Aldrich</td><td>M2643</td><td></td></tr><tr><td>Ammonium chloride</td><td>Sigma Aldrich</td><td>A9434</td><td></td></tr><tr><td>Tris-base</td><td></td><td></td><td></td></tr><tr><td>Dimethyl Sulfoxide</td><td>Sigma Aldrich&amp;nbsp;</td><td>D8418</td><td></td></tr><tr><td>(5-(and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE)</td><td>Molecular Probes</td><td>C-1157</td><td>Reconstitute in DMSO</td></tr><tr><td>Phorbol 12,13-dibutyrate (PBDU, Phorbol ester)</td><td>Sigma Aldrich</td><td>P1269</td><td></td></tr><tr><td>A23187 (Calcium ionophore)</td><td>Sigma Aldrich</td><td>C7522</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Antibodies and recombinant protein&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>CD11b biotin (clone m1/70)</td><td>Biolegend</td><td>101204</td><td>T cell depletion cocktail</td></tr><tr><td>CD11c biotin (clone N418)</td><td>Biolegend</td><td>117304</td><td>T cell depletion cocktail</td></tr><tr><td>Gr-1 biotin (clone RB6-8C5)</td><td>Biolegend</td><td>108404</td><td>T cell depletion cocktail</td></tr><tr><td>Ter119 biotin (clone Ter119)</td><td>Biolegend</td><td>116204</td><td>T cell depletion cocktail</td></tr><tr><td>TCR-&amp;gamma;&amp;delta; biotin (clone GL-3)</td><td>Biolegend</td><td>118103</td><td>T cell depletion cocktail</td></tr><tr><td>CD19 biotin (clone 6D5)</td><td>Biolegend</td><td>115504</td><td>T cell depletion cocktail</td></tr><tr><td>B220 biotin (clone RA3-6B2)</td><td>Biolegend</td><td>103204</td><td>T cell depletion cocktail</td></tr><tr><td>CD49b biotin (clone DX5)</td><td>Biolegend</td><td>108904</td><td>T cell depletion cocktail</td></tr><tr><td>CD4 biotin (clone GK1.5)</td><td>Biolegend</td><td>100404</td><td>T cell depletion cocktail</td></tr><tr><td>CD8 biotin (clone 53-6.7)</td><td>Biolegend</td><td>100704</td><td>T cell depletion cocktail</td></tr><tr><td>F(ab&amp;rsquo;)2 goat anti-mouse IgM (plate coated)</td><td>Jackson ImmunoResearch&amp;nbsp;</td><td>115-006-075</td><td>50 &amp;micro;l/well for coating (96-well)</td></tr><tr><td>Anti-mouse CD40 mAb (plate coated)</td><td>Pharmingen&amp;nbsp;</td><td>553722</td><td>50 &amp;micro;l/well for coating (96-well)</td></tr><tr><td>Recombinant IL-4</td><td>ProSpec&amp;nbsp;</td><td>Cyt-282</td><td></td></tr><tr><td>LPS from E. coli Serotype 055:B5</td><td>Sigma Aldrich</td><td>L-4005</td><td></td></tr><tr><td>Anti-CD3 (clone clone OKT3) (plate coated)</td><td>eBioscience&amp;nbsp;</td><td>16-0037-85</td><td>50 &amp;micro;l/well for coating (96-well)</td></tr><tr><td>Anti-CD28 (clone clone 37.51) (plate coated)</td><td>eBioscience&amp;nbsp;</td><td>16-0281-85</td><td>50 &amp;micro;l/well for coating (96-well)</td></tr><tr><td>Recombinant IL-2</td><td>ProSpec</td><td>Cyt-370</td><td></td></tr><tr><td>Albumin from chicken egg white, Ovalbumin</td><td>Sigma Aldrich</td><td>A7641</td><td></td></tr></tbody></table>

isolation and activation of murine lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Magnetzellreinigung von Lymphozyten erlaubt Benutzern, eine Zielzellpopulation in einer relativ kurzen Zeit zu reinigen. Mit unserem Verarmungs Protokoll konnten wir den Prozentsatz der CD8-T-Zellen (OT-I in Rekombinations-aktivierendes Gen-1 (RAG-1) -defiziente Mäuse) von 72,8% (vor Reinigung) auf 94,2% (nach der Reinigung zu erhöhen; 1A) 4,5. Diese gereinigten Lymphozyten können dann für die nachfolgende funktionelle Assays verwendet werden , um die Lymp...

Watch this Scientific Journal Video about Isolation and Activation of Murine Lymphocytes at JoVE.com

Isolation and Activation of Murine Lymphocytes

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

Isolation und Aktivierung von murinen Lymphozyten

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

isolation-and-activation-of-murine-lymphocytes

Research

JoVE Journal

Immunology and Infection

21.0K Aufrufe.  Nanyang Technological University. Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

Serie: Isolation and Activation of Murine Lymphocytes

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

Mausmodell der CD40-Aktivierung von B-Zellen

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

Forschung

Immunologie und Infektion

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

Retrovirale Transduktion von T-Zell-Rezeptoren in Maus-T-Zellen

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

Dendritic cells (DC) are heterogeneous cell populations that differ in their cell membrane markers, migration patterns and distribution, and in their antigen presentation and T cell activation capacities. Since most vaccinations of experimental tumor models require millions of DC, they are widely isolated from the bone marrow or spleen. However, these DC significantly differ from blood and tumor DC in their responses to immune complexes (IC), and presumably to other Syk-coupled lectin receptors. Importantly, given the sensitivity of DC to danger-associated molecules, the presence of endotoxins or antibodies that crosslink activation receptors in one of the isolating steps could result in the priming of DC and thus affect the parameters, or at least the dosage, required to activate them. Therefore, here we describe a detailed protocol for isolating MoDC from blood and tumors while avoiding their premature activation. In addition, a protocol is provided for MoDC activation with tumor IC, and their subsequent analyses.

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

Monocyte-derived DC (MoDC) can sense minor amounts of danger-associated molecules and are therefore easily primed. We provide a detailed protocol for the isolation of MoDC from blood and tumors and their activation with immune complexes while highlighting key precautions that should be considered in order to avoid their premature activation.

Isolierung-Protokoll der Maus Monocyte-abgeleitete Dendritische Zellen und ihre anschließende In-vitro- Aktivierung mit Tumor Immunkomplexe

Dendritic cells (DC) are heterogeneous cell populations that differ in their cell membrane markers, migration patterns and distribution, and in their ...

Krebsforschung

Isolation of CD4+ T cells from Mouse Lymph Nodes Using Miltenyi MACS Purification

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several organisms including humans, non-human primates, rat and, as we describe here, mice. Magnetic bead-based cell separation allows for either positive selection (or cell depletion) as well as negative selection. Here, we demonstrate negative selection of untouched or na ve CD4+ helper T cells. Using this standard protocol we typically purify cells that are &#8805; 96% pure CD4+/CD3+. This protocol is used in conjunction with the protocol Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice published in issue 7 of JoVE, for purification of T cells and other cell types to adoptively transfer for imaging purposes. Although we did not demonstrate FACS analysis in this protocol video, it is highly recommended to check the overall purity of isolated cells using the appropriate antibodies via FACS. In addition, we demonstrate the non-sterile method of T cell isolation. If sterile cells are needed for your particular end-user application, be sure to do all of the demonstrated procedures in the tissue culture hood under standard sterile conditions. Thank you for watching and good luck with your own experiments!

Isolation of lymphocytes using the Miltenyi MACs kit is a reliable way to purify cells from whole lymphoid tissue homogenates. Cells purified using the Miltenyi system are typically &#8805; 96% pure. Here, we demonstrate the steps taken to isolate CD4+ T cells, one of the many kits offered by Miltenyi.

Isolierung von CD4 + T-Zellen aus Maus-Lymphknoten mit Miltenyi MACS Purification

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several ...

Biologie

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and bacteria 1. Mucosae are also inductive sites in the host to generate immunity against pathogens, such as the Peyers patches in the intestinal tract and the nasal-associated lymphoreticular tissue in the respiratory tract. This unique feature brings mucosal immunity as a crucial player of the host defense system. Many studies have been focused on gastrointestinal and respiratory mucosal sites. However, there has been little investigation of reproductive mucosal sites. The genital tract mucosa is the primary infection site for sexually transmitted diseases (STD), including bacterial and viral infections. STDs are one of the most critical health challenges facing the world today. Centers for Disease Control and Prevention estimates that there are 19 million new infectious every year in the United States. STDs cost the U.S. health care system $17 billion every year 2, and cost individuals even more in immediate and life-long health consequences. In order to confront this challenge, a greater understanding of reproductive mucosal immunity is needed and isolating lymphocytes is an essential component of these studies. Here, we present a method to reproducibly isolate lymphocytes from murine female genital tracts for immunological studies that can be modified for adaption to other species. The method described below is based on one mouse.&#x2003;

An efficient way to isolate lymphocytes from mouse genital tract is described. This method takes advantage of enzyme digestion and Percoll gradient separation to allow efficient isolation. This technique is also adaptable to for use in other species

Isolierung von Lymphozyten aus Maus Genital Tract Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and ...

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 of Murine Lymph Node Stromal Cells

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host. 
Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.

Isolation of lymph node stromal cells is a multistep procedure including enzymatic digestion and mechanical disaggregation to obtain fibroblastic reticular cells, lymphatic and blood endothelial cells. In the described procedure, a short digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells.

Isolierung von murinen Lymphknoten Stromazellen

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and ...

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen presenting molecules to initiate T-cell-mediated immunity. Dendritic cells can be separated into several phenotypically and functionally heterogeneous subsets. Three important subsets of splenic dendritic cells are plasmacytoid, CD8&#945;Pos and CD8&#945;Neg cells. The plasmacytoid DCs are natural producers of type I interferon and are important for anti-viral T cell immunity. The CD8&#945;Neg DC subset is specialized for MHC class II antigen presentation and is centrally involved in priming CD4 T cells. The CD8&#945;Pos DCs are primarily responsible for cross-presentation of exogenous antigens and CD8 T cell priming. The CD8&#945;Pos DCs have been demonstrated to be most efficient at the presentation of glycolipid antigens by CD1d molecules to a specialized T cell population known as invariant natural killer T (iNKT) cells. Administration of Flt-3 ligand increases the frequency of migration of dendritic cell progenitors from bone marrow, ultimately resulting in expansion of dendritic cells in peripheral lymphoid organs in murine models. We have adapted this model to purify large numbers of functional dendritic cells for use in cell transfer experiments to compare in vivo proficiency of different DC subsets.

Distinct dendritic cell subsets exist as rare populations in lymphoid organs, and therefore are challenging to isolate in sufficient numbers and purity for immunological experiments. Here we describe a high efficiency, high yield method for isolation of all of the currently known major subsets of mouse splenic dendritic cells.

Eine effiziente und High-Yield-Methode zur Isolierung von Maus über dendritische Zellen Subsets

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

Isolieren von Lymphozyten aus der Maus kleinen intestinalen Immunsystems

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...

Isolierung von Endothelzellen aus dermalen Lymphkapillaren der Maus

Murine Dermal Lymphatic Endothelial Cell Isolation

The lymphatic system participates in the regulation of immune surveillance, lipid absorption, and tissue fluid balance. The isolation of murine lymphatic endothelial cells is an important process for lymphatic research, as it allows the performance of in vitro and biochemical experiments on the isolated cells. Moreover, the development of Cre-lox technology has enabled the tissue-specific deficiency of genes that cannot be globally targeted, leading to the precise determination of their role in the studied tissues. The dissection of the role of certain genes in lymphatic physiology and pathophysiology requires the use of lymphatic-specific promoters, and thus, the experimental verification of the expression levels of the targeted genes.
Methods for efficient isolation of lymphatic endothelial cells from wild-type or transgenic mice enable the use of ex vivo and in vitro assays to study the mechanisms regulating the lymphatic functions and the identification of the expression levels of the studied proteins. We have developed, standardized and present a protocol for the efficient isolation of murine dermal lymphatic endothelial cells (DLECs) via magnetic bead purification based on LYVE-1 expression. The protocol outlined aims to equip researchers with a tool to further understand and elucidate important players of lymphatic endothelial cell functions, especially in facilities where fluorescence-activated cell sorting equipment is not available.

Autor im Rampenlicht: Magnetische Bead-basierte Isolierung von murinen dermalen lymphatischen Endothelzellen

This paper describes a method for magnetic bead-based isolation of murine endothelial cells from dermal lymphatic capillaries. The isolated lymphatic endothelial cells can be used for downstream in vitro experiments and protein expression analysis.

Isolierung von dermalen lymphatischen Endothelzellen der Maus

The lymphatic system participates in the regulation of immune surveillance, lipid absorption, and tissue fluid balance. The isolation of murine lymphatic ...