Diese Arbeit wurde durch die Smith Family Award for Excellence in Biomedical Research und NIH RO1 AI083426-01 (FW) gefördert. Patrick Maschmeyer und Melanie Flach von PhD-Stipendien des Boehringer Ingelheim-Stiftung unterstützt.

C57BL / 6 Mäusen sollte bei ~ 20 Wochen alt oder älter verwendet werden. Die Verwendung von männlichen Mäusen mit einem Gewicht von 25-30g wird empfohlen. Die Ausbeute an Sternzellen kann durch die Fütterung Mäusen ein Vitamin A angereicherte Diät für 2 Monate vor Zellisolation Sternzellen erhöht werden. Etwa 2x10 5 Lebersternzellen kann aus der Leber von einem C57BL / 6 Maus gereinigt werden, während die Ausbeute der Sternzellen aus Balb / c Mäusen deutlich höher. Das folgende Protokoll ist auf 5 Mäusen angepasst. Tierpflege und Experimente wurden in Übereinstimmung mit genehmigten Institutional Animal Care und Verwenden Ausschuss-Protokolle durchgeführt. 1. In situ Perfusion der Maus Leber mit Verdauungsenzymen <ol><li> Warm die SC1 Puffer und Enzym Perfusionslösungen in einem 37 ° C Wasserbad. </li><li> Montieren Sie einen geflügelten Infusion auf den Silikonschlauch einer Schlauchpumpe gesetzt. </li><li> Kalibrieren Sie die Pumpe mit PBS zu einer laminaren Strömung der 6.5ml/min, die den Durchfluss, die für die in situ Perfusion der Leber verwendet wird, ist zu erhalten. Äquilibrieren der Silikonschlauch mit SC1-Lösung. </li><li> Anesthetize der Maus durch intraperitoneale Injektion von Ketamin (90 mg / kg) und Xylazin (10 mg / kg) Lösung und testen für den Verlust von Reflexen, tiefe Betäubung zu gewährleisten. </li><li> Befestigen Sie die Maus in Rückenlage auf einer geeigneten Unterlage. </li><li> Mit einer Schere und Pinzette, um einen Längsschnitt in der Bauchhaut führen und setzen das Bauchfell. </li><li> Öffnen Sie vorsichtig das Bauchfell und bewegen Sie den Darm aus der Bauchhöhle auf die linke Seite des Tieres, um die Pfortader aussetzen. </li><li> Legen Sie die Kanüle der Infusion in die Pfortader gesetzt. Für diesen Schritt ist die Verwendung von einem Stereomikroskop empfohlen. </li><li> Starten Sie die Durchblutung der Leber mit 30ml SC1-Lösung. Öffnen Sie die Vena cava inferior unmittelbar nach Beginn der Strömung. Erfolgreiche Spülung der Leber wird durch einen Verlust der Farbe des Lebergewebes angegeben. </li><li> Perfundieren der Leber mit 30 ml Pronase E Lösung. Nach erfolgreicher Verdauung wird die Leberlappen schwellen an, und die Läppchen erscheinen verschiedene durch die Kapsel. </li><li> Perfundieren der Leber mit 30 ml Collagenase P-Lösung. Die Leber an diesem Punkt hat seine Form verloren und sieht atonische und amorph. </li><li> Sorgfältig trennen die Leber vom Zwerchfell und den umliegenden Organen und speichern Sie es in 70ml SC2-Lösung auf Eis. </li><li> Wiederholen Sie den Vorgang für weitere Mäuse. </li></ol> 2. In-vitro-Verdauung der Maus Leber Alle weiteren Arbeitsschritte sind unter sterilen Bedingungen in einer Sterilbank durchgeführt werden. <ol><li> Schneiden Sie die Leber in Stücke von ca. 2x2x2mm 3 mit einer scharfen Schere. </li><li> Kombinieren Sie die Aussetzung der 70ml SC2 einschließlich der Leber Stücke mit 50 ml Pronase E-Collagenase P-Lösung und 1ml von DNase I-Lösung. </li><li> Digest der Leber für 20min bei 37 ° C unter Rühren. </li></ol> 3. Dichtegradientenzentrifugation <ol><li> Filtern Sie die Zellsuspension durch 70μm Zelle Siebe in 6 50ml Falcon-Röhrchen und bis zu 50ml mit SC2-Puffer. Zentrifuge für 10 min bei 600 g und 4 ° C. </li><li> Vorsichtig absaugen 40ml der Überstand, fügen Sie dann 150 &amp;mgr; l DNase I-Lösung in jedes Röhrchen und Zellen. </li><li> Pool der Zellsuspensionen in 4 50ml Falcon-Röhrchen und wäscht mit GBSS-B, Zentrifuge für 10 min bei 600 g und 4 ° C. </li><li> Vorsichtig absaugen so viel von der Überstand wie möglich, ohne das Pellet und fügen 150 &amp;mgr; l DNase I-Lösung in jedes Röhrchen. Resuspendieren der Zellpellets in 10ml GBSS-B pro Röhre. </li><li> Pool der Zellen in 2 50ml Falcon-Röhrchen und fügen GBSS-B auf ein Gesamtvolumen von 36ml pro Tube. Fügen Sie 14ml Nycodenz Lösung in jedes Röhrchen und gut mischen. </li><li> Transfer-10ml der Zellsuspension in ein 12ml Gradientenzentrifugation Röhre, die sich in 10 Röhren in total. Gently überlagern die Zellsuspension mit 1,5 ml GBSS-B pro Röhre. </li><li> Centrifuge die Gradienten für 15 min bei 1500g und 4 ° C ohne Bremse. Anschließend wird Hepatozyten werden am Boden des Röhrchens pelletiert während sternförmigen Zellen in der Interphase als weißer Ring zu finden sind. </li><li> Sorgfältig Ernte der Interphase mit den sternförmigen Zellen und waschen Sie sie mit GBSS-B; Zentrifuge für 10 min bei 600 g und 4 ° C. </li><li> Saugen Sie den Überstand und Zellen in 20ml DMEM mit 10% Hitze-inaktiviertes FBS, 1% Penicillin / Streptomycin, 2 mM L-Glutamin, 1 mM Natriumpyruvat, und 10mm HEPES ergänzt. Übertragen Sie die Zellen in Zellkulturflaschen mit einer Konzentration von 2x10 4 Zellen / cm 2. Inkubieren Sie die Zellen bei 37 ° C und 5% CO 2. </li><li> Ändern Sie die Medien so schnell wie der hepatischen Sternzellen Anhänger (ca. 2h nach der Herstellung) abzuwaschen toten Zellen und Zelltrümmer werden. </li><li> Am folgenden Tag, dem sternförmigen Zellens sollten ihre charakteristischen sternförmigen Morphologie mit perinukleären Vitamin A-Speicherung Lipidvesikel. </li><li> Für nachfolgende Experimente können Lebersternzellen leicht von nicht-beschichteten Kunststoff-Oberflächen mit einer milden enzymatischen Lösungen wie Accutase, zB gelöst werden </li></ol> 4. Repräsentative Ergebnisse: Nach der Vorbereitung der hepatischen Sternzellen mit dem Protokoll vorgesehen ist, kann die Reinheit der isolierten Population getestet erwägt drei wichtigsten Merkmale dieser Zelltyp, wie etwa sternförmig, perinukleären Fetttröpfchen, und die Expression von sauren Gliafaserproteins (GFAP werden ). Vertreter Bilder für das charakteristische Aussehen der hepatischen Sternzellen 2h nach Zellisolation, sowie am Tag 1 und 3 der in-vitro-Kultur sind in Abbildung 1 dargestellt. Abbildung 2 zeigt eine repräsentative Immunfluoreszenzfärbung für GFAP in hepatischen Sternzellen, die für 3 Tage nach der Zell-Isolierung wurden kultiviert. Lebersternzellen differenzieren sich in myofibroblastischer Zellen während der in vitro-Kultur. Ein charakteristisches Merkmal dieser aktiviert Sternzellen ist der Ausdruck von alpha smooth muscle actin (αSMA). Abbildung 3 zeigt Immunfluoreszenzfärbung für Asma und Myosin IIA in aktivierten Sternzellen am Tag 7 der in vitro-Kultur. <img src="/files/ftp_upload/2710/2710fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Charakteristischer Morphologie von hepatischen Sternzellen. Sternzellen wurden von der Maus Leber unter Verwendung des Protokolls zur Verfügung gestellt isoliert. Die Bilder zeigen Sternzellen 2h nach Zellisolation (a, b), sowie am Tag 1 (c) und Tag 3 (d) der in vitro-Kultur. Hepatische Sternzellen weisen eine hohe Mengen an Lipid-Vesikel an perinukleären Websites und erhalten ihre unverwechselbare astral-Morphologie in den ersten Tagen der in vitro-Kultur (Vergrößerung 200x). <img src="/files/ftp_upload/2710/2710fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Lebersternzellen speziell ausdrückliche GFAP in der Leber. Stellate Zellen wurden isoliert und immunofluorescently für GFAP (rot) gefärbt am Tag 3 der In-vitro-Kultur. Zellkerne sind blau (Hoechst-Färbung) dargestellt. <img src="/files/ftp_upload/2710/2710fig3.jpg" alt="Abbildung 3" /> Abbildung 3. Liver Sternzellen differenzieren sich in Myofibroblasten. Hepatische Sternzellen aus C57BL / 6 Mäusen isoliert wurden für 7 Tage kultiviert. Anschließend wurden sie an die Kammer Dias übertragen und gefärbt Asma (rot dargestellt) und Myosin IIA (dargestellt in Grün). Die Zellkerne wurden mit Hoechst (blau) gegengefärbt. <table border="1"><tbody><tr><td colspan="2"> SC1 Buffer </td></tr><tr><td> EGTA </td><td> 190 mg </td></tr><tr><td> Glucose </td><td> 900mg </td></tr><tr><td> HEPES </td><td> 10 ml 1 M Stammlösung </td></tr><tr><td> KCl </td><td> 400mg </td></tr><tr><td> Na 2 HPO 4 x 2 H 2 O </td><td> 151mg </td></tr><tr><td> NaCl </td><td> 8g </td></tr><tr><td> NaH 2 PO 4 x H 2 O </td><td> 78mg </td></tr><tr><td> NaHCO 3 </td><td> 350mg </td></tr><tr><td> Phenol Red </td><td> 6mg </td></tr><tr><td> dH 2 O </td><td> auffüllen auf 1l </td></tr><tr><td colspan="2"> SC2 Buffer </td></tr><tr><td> CaCl 2 x 2H 2 O </td><td> 560mg </td></tr><tr><td> HEPES </td><td> 10 ml 1M Stammlösung </td></tr><tr><td> KCl </td><td> 400mg </td></tr><tr><td> Na 2 HPO 4 x 2 H 2 O </td><td> 151mg </td></tr><tr><td> NaCl </td><td> 8g </td></tr><tr><td> NaH 2 PO 4 x H 2 O </td><td> 78mg </td></tr><tr><td> NaHCO 3 </td><td> 350mg </td></tr><tr><td> Phenol Red </td><td> 6mg </td></tr><tr><td> dH 2 O </td><td> auffüllen auf 1l </td></tr><tr><td colspan="2"> GBSS-A-Puffer </td></tr><tr><td> KCl </td><td> 370mg </td></tr><tr><td> CaCl 2 x 2H 2 O </td><td> 225mg </td></tr><tr><td> Glucose </td><td> 991mg </td></tr><tr><td> KH 2 PO 4 </td><td> 30mg </td></tr><tr><td> MgCl 2 x 6 H 2 O </td><td> 210mg </td></tr><tr><td> MgSO 4 x 7 H 2 O </td><td> 70mg </td></tr><tr><td> Na 2 HPO 4 x 2 H 2 O </td><td> 75mg </td></tr><tr><td> NaHCO 3 </td><td> 227mg </td></tr><tr><td> Phenol Red </td><td> 6mg </td></tr><tr><td> dH 2 O </td><td> auffüllen auf 1l </td></tr><tr><td colspan="2">GBSS-B Buffer </td></tr><tr><td> CaCl 2 x 2H 2 O </td><td> 225mg </td></tr><tr><td> Glucose </td><td> 991mg </td></tr><tr><td> KCl </td><td> 370mg </td></tr><tr><td> KH 2 PO 4 </td><td> 30mg </td></tr><tr><td> MgCl 2 x 6 H 2 O </td><td> 210mg </td></tr><tr><td> MgSO 4 x 7 H 2 O </td><td> 70mg </td></tr><tr><td> Na 2 HPO 4 x 2 H 2 O </td><td> 75mg </td></tr><tr><td> NaCl </td><td> 8g </td></tr><tr><td> NaHCO 3 </td><td> 227mg </td></tr><tr><td> Phenol Red </td><td> 6mg </td></tr><tr><td> dH 2 O </td><td> auffüllen auf 1l </td></tr><tr><td colspan="2"> Pronase E Perfusionslösung </td></tr><tr><td> Pronase E </td><td> 100mg (4000 PU / mg min) </td></tr><tr><td> SC2 Buffer </td><td> 200ml </td></tr><tr><td colspan="2"> Collagenase P Perfusionslösung </td></tr><tr><td> Collagenase P </td><td> 85mg (1,78 U / mg lyo) </td></tr><tr><td> SC2 Buffer </td><td> 200ml </td></tr><tr><td colspan="2"> Pronase E-Collagenase P-Lösung </td></tr><tr><td> Pronase E </td><td> 50mg (4000 PU / mg min) </td></tr><tr><td> Collagenase P </td><td> 85mg (1,78 U / mg lyo) </td></tr><tr><td> SC2 Buffer </td><td> 50ml </td></tr><tr><td colspan="2"> DNase I-Lösung </td></tr><tr><td> DNase I </td><td> 6mg (ca. 2000U/mg) </td></tr><tr><td> GBSS-B </td><td> 3ml </td></tr><tr><td colspan="2"> Nycodenz Lösung </td></tr><tr><td> Nycodenz </td><td> 8g </td></tr><tr><td> GBSS-A </td><td> 28ml </td></tr></tbody></table> Tabelle 1. Puffer und Enzym-Lösungen für die Isolierung von hepatischen Sternzellen erforderlich. Der pH-Wert aller Puffer sollte 7,3-7,4 eingestellt werden. Darüber hinaus ist Sterilfiltration aller Puffer empfohlen. Es ist wichtig, die Menge des Enzyms, die gemäß der gegebenen enzymatischen Aktivität anzupassen.

<ol>
	<li>Friedman, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+stellate+cells:+protean,+multifunctional,+and+enigmatic+cells+of+the+liver.">Hepatic stellate cells: protean, multifunctional, and enigmatic cells of the liver.</a> Physiol Rev. 88, 125-172 (2008).</li><li>Geerts, A. H. i. s. t. o. r. y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=heterogeneity,+developmental+biology,+and+functions+of+quiescent+hepatic+stellate+cells.">heterogeneity, developmental biology, and functions of quiescent hepatic stellate cells.</a> Semin Liver Dis. 21, 311-335 (2001).</li><li>Gressner, A. M. &. a. m. p. ;. a. m. p., Weiskirchen, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modern+pathogenetic+concepts+of+liver+fibrosis+suggest+stellate+cells+and+TGF-beta+as+major+players+and+therapeutic+targets.">Modern pathogenetic concepts of liver fibrosis suggest stellate cells and TGF-beta as major players and therapeutic targets.</a> J Cell Mol Med. 10, 76-99 (2006).</li><li>Reynaert, H., Thompson, M. G., Thomas, T., Geerts, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+stellate+cells:+role+in+microcirculation+and+pathophysiology+of+portal+hypertension.">Hepatic stellate cells: role in microcirculation and pathophysiology of portal hypertension.</a> Gut. 50, 571-581 (2002).</li><li>Winau, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ito+cells+are+liver-resident+antigen-presenting+cells+for+activating+T+cell+responses.">Ito cells are liver-resident antigen-presenting cells for activating T cell responses.</a> Immunity. 26, 117-129 (2007).</li><li>Minicis, S. D. e. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+profiles+during+hepatic+stellate+cell+activation+in+culture+and+in+vivo.">Gene expression profiles during hepatic stellate cell activation in culture and in vivo.</a> Gastroenterology. 132, 1937-1946 (2007).</li><li>Knittel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+liver+myofibroblasts+and+hepatic+stellate+cells+in+normal+and+diseased+rat+livers:+distinct+roles+of+(myo-)fibroblast+subpopulations+in+hepatic+tissue+repair.">Localization of liver myofibroblasts and hepatic stellate cells in normal and diseased rat livers: distinct roles of (myo-)fibroblast subpopulations in hepatic tissue repair.</a> Histochem Cell Biol. 112, 387-401 (1999).</li></ol>

Lebersternzellen regeln wesentliche physiologische und pathophysiologische Prozesse in der Leber. Darüber hinaus besitzen Sternzellen Antigen-präsentierenden Eigenschaften, wodurch sie ein wichtiger Bestandteil der Leber Immunantwort. Obwohl Lebersternzellen 10-15% der gesamten Zellzahl in der Leber enthalten, ist die Isolation dieser Zellen Herausforderung aufgrund ihrer Lokalisation in der perisinusoidal Raum der Disse. Hier präsentieren wir Ihnen eine einfache Methode, um Lebersternzel...

<table border="1"><tbody><tr><th> Name Of Reagent </th><th> Firma </th><th> Katalog Nr. </th></tr><tr><td> 70μm Zellsieb </td><td> BD </td><td> 352350 </td></tr><tr><td> CaCl 2 x 2H 2 O </td><td> Sigma-Aldrich </td><td> C3306 </td></tr><tr><td> Collagenase P </td><td> Roche </td><td> 11249002001 </td></tr><tr><td> DMEM </td><td> Gibco </td><td> 11960 </td></tr><tr><td> DNase I </td><td> Roche </td><td> 10104159001 </td></tr><tr><td> DPBS </td><td> CellGro </td><td> 21 bis 031-cv </td></tr><tr><td> EGTA </td><td> Fluka </td><td> 3777 </td></tr><tr><td> Fetal Bovine Serum </td><td> Gibco </td><td> 16000 </td></tr><tr><td> Glucose </td><td> Sigma-Aldrich </td><td> G7528 </td></tr><tr><td> Gradientenzentrifugation Tubes </td><td> Greiner Bio-One </td><td> 163160 </td></tr><tr><td> HEPES </td><td> Gibco </td><td> 15630 </td></tr><tr><td> KCL </td><td> Sigma-Aldrich </td><td> P9541 </td></tr><tr><td> KH 2 PO 4 </td><td> Sigma-Aldrich </td><td> P9791 </td></tr><tr><td> L-Glutamin </td><td> Gibco </td><td> 25030 </td></tr><tr><td> MgCl 2 x 6 H 2 O </td><td> Fluka </td><td> 63068 </td></tr><tr><td> MgSO 4 x 7H 2 O </td><td> Sigma-Aldrich </td><td> M2773 </td></tr><tr><td> Na 2 HPO 4 x 2H 2 O </td><td> Fluka </td><td> 71643 </td></tr><tr><td> NaCl </td><td> Sigma-Aldrich </td><td> S3014 </td></tr><tr><td> NaH 2 PO 4 x H 2 O </td><td> Fluka </td><td> 71507 </td></tr><tr><td> NaHCO 3 </td><td> Fluka </td><td> 71628 </td></tr><tr><td> Nycodenz </td><td> Accudenz AG </td><td> AN7050/BLK </td></tr><tr><td> Penicillin / Streptomycin </td><td> Gibco </td><td> 15140 </td></tr><tr><td> Schlauchpumpen </td><td> Cole Parmer </td><td> HV-7523 bis 70 </td></tr><tr><td> Phenol Red </td><td> Sigma-Aldrich </td><td> P4633 </td></tr><tr><td> Pronase E </td><td> Calbiochem </td><td> 7433-2 </td></tr><tr><td> Silikonschlauch </td><td> Masterflex </td><td> HV-96440-14 </td></tr><tr><td> Natriumpyruvat </td><td> Gibco </td><td> 11360 </td></tr><tr><td> Zellkulturflasche (25cm 2) </td><td> BD </td><td> 353108 </td></tr><tr><td> Winged Infusion Set </td><td> Terumo </td><td> 1SV27EL </td></tr></tbody></table>

seven steps to stellate cells

Hepatische Sternzellen sind Leber-residenten Zellen des sternförmigen Morphologie und sind in den Raum der Disse zwischen Leber sinusförmige Endothelzellen und Hepatozyten 1,2 liegt. Sternzellen sind aus dem Knochenmark Vorstufen abgeleitet und bis zu 80% der gesamten Körper Vitamin A 1, 2. Nach der Aktivierung, Differenzierung Sternzellen in Myofibroblasten um extrazelluläre Matrix produzieren und damit einen Beitrag zur Leberfibrose 3. Auf der Grundlage ihrer Fähigkeit zur Kontraktion kann myofibroblastischer Sternzellen regulieren den Gefäßtonus mit portaler Hypertonie 4 zugeordnet. Kürzlich haben wir gezeigt, dass Lebersternzellen sind potente Antigen-präsentierende Zellen und können NKT-Zellen als auch konventionelle T-Lymphozyten 5 zu aktivieren. Hier präsentieren wir eine Methode zur effizienten Erstellung von hepatischen Sternzellen aus Maus-Leber. Aufgrund ihrer perisinusoidal Lokalisation, ist die Isolierung von hepatischen Sternzellen in einem mehrstufigen Prozess. Um Sternzellen zugänglich Isolierung aus dem Raum der Disse machen, Maus Leber in situ mit den Verdauungsenzymen Pronase E und Collagenase P. Nach Perfusion perfundiert werden, wird das Lebergewebe, um zusätzliche enzymatische Behandlung mit Pronase E und Collagenase P in unterzogen vitro. Anschließend findet die Methode den Vorteil der großen Menge an Vitamin A-Speicherung Fetttröpfchen in hepatischen Sternzellen. Diese Funktion ermöglicht die Trennung von sternförmigen Zellen aus anderen Leber Zelltypen durch Zentrifugation auf einem 8% Nycodenz Gradienten. Die hier beschriebene Protokoll ergibt sich eine sehr reine und homogene Population von Sternzellen. Die Reinheit der Präparate können durch Färbung für die Marker-Molekül sauren Gliafaserproteins (GFAP), vor der Analyse mittels Fluoreszenzmikroskopie oder Durchflusszytometrie beurteilt werden. Zeigt ferner, wie die Lichtmikroskopie das einzigartige Erscheinungsbild der sternförmigen Lebersternzellen, dass große Mengen von Fetttröpfchen Hafen. Zusammen bilden wir ein detailliertes Protokoll für die effiziente Isolierung von hepatischen Sternzellen, darunter repräsentative Bilder ihrer morphologischen Erscheinungsbild und GFAP Ausdruck, der den sternförmigen Zellen Einheit definieren helfen.

Watch this Scientific Journal Video about Seven Steps to Stellate Cells at JoVE.com

Seven Steps to Stellate Cells

Hier beschreiben wir eine Methode zur Isolierung von hepatischen Sternzellen aus Maus-Leber. Für Sternzellen Zellreinigung werden Maus-Leber verdaut<em&gt; In situ</em&gt; Und<em&gt; In-vitro-</em&gt; Nach Pronase-Kollagenase-Behandlung vor der Dichtegradientenzentrifugation. Diese Technik liefert hochreine Lebersternzellen.

Seven Steps to Cells Stellate

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

Research

JoVE Journal

Immunology and Infection

33.5K Aufrufe.  Harvard Medical School. Hier beschreiben wir eine Methode zur Isolierung von hepatischen Sternzellen aus Maus-Leber. Für Sternzellen Zellreinigung werden Maus-Leber verdaut In situ Und In-vitro- Nach Pronase-Kollagenase-Behandlung vor der Dichtegradientenzentrifugation. Diese Technik liefert hochreine Lebersternzellen.

Serie: Seven Steps to Stellate Cells

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of guard cells, which are able to close the stomatal pore in response to a number of external factors including light intensity, carbon dioxide concentration, and relative humidity (RH). The stomatal pore is also the main route for pathogen entry into leaves, a crucial step for disease development. Recent studies have unveiled that closure of the pore is effective in minimizing bacterial disease development in Arabidopsis plants; an integral part of plant innate immunity. Previously, we have used epidermal peels to assess stomatal response to live bacteria (Melotto et al. 2006); however maintaining favorable environmental conditions for both plant epidermal peels and bacterial cells has been challenging. Leaf epidermis can be kept alive and healthy with MES buffer (10 mM KCl, 25 mM MES-KOH, pH 6.15) for electrophysiological experiments of guard cells. However, this buffer is not appropriate for obtaining bacterial suspension. On the other hand, bacterial cells can be kept alive in water which is not proper to maintain epidermal peels for long period of times. When an epidermal peel floats on water, the cells in the peel that are exposed to air dry within 4 hours limiting the timing to conduct the experiment. An ideal method for assessing the effect of a particular stimulus on guard cells should present minimal interference to stomatal physiology and to the natural environment of the plant as much as possible. We, therefore, developed a new method to assess stomatal response to live bacteria in which leaf wounding and manipulation is greatly minimized aiming to provide an easily reproducible and reliable stomatal assay. The protocol is based on staining of intact leaf with propidium iodide (PI), incubation of staining leaf with bacterial suspension, and observation of leaves under laser scanning confocal microscope. Finally, this method allows for the observation of the same live leaf sample over extended periods of time using conditions that closely mimic the natural conditions under which plants are attacked by pathogens.

We have developed a simple and reproducible protocol to access stomatal response to live bacteria. This method minimizes wounding and manipulation of the leaf as compared to the use of epidermal peels reported previously.

Die Beurteilung stomatäre Response to Bakterienzellen Live mit Whole Leaf Imaging

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of ...

Forschung

Immunologie und Infektion

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

Nach Cell-Schicksal<em&gt; E. coli</em&gt; Nach Infektion mit Phagen

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

A Modified EPA Method 1623 that Uses Tangential Flow Hollow-fiber Ultrafiltration and Heat Dissociation Steps to Detect Waterborne Cryptosporidium and Giardia spp.

Cryptosporidium and Giardia species are two of the most prevalent protozoa that cause waterborne diarrheal disease outbreaks worldwide. To better characterize the prevalence of these pathogens, EPA Method 1623 was developed and used to monitor levels of these organisms in US drinking water supplies 12. The method has three main parts; the first is the sample concentration in which at least 10 L of raw surface water is filtered. The organisms and trapped debris are then eluted from the filter and centrifuged to further concentrate the sample. The second part of the method uses an immunomagnetic separation procedure where the concentrated water sample is applied to immunomagnetic beads that specifically bind to the Cryptosporidium oocysts and Giardia cysts allowing for specific removal of the parasites from the concentrated debris. These (oo)cysts are then detached from the magnetic beads by an acid dissociation procedure. The final part of the method is the immunofluorescence staining and enumeration where (oo)cysts are applied to a slide, stained, and enumerated by microscopy.
Method 1623 has four listed sample concentration systems to capture Cryptosporidium oocysts and Giardia cysts in water: Envirochek filters (Pall Corporation, Ann Arbor, MI), Envirochek HV filters (Pall Corporation), Filta-Max filters (IDEXX, Westbrook, MA), or Continuous Flow Centrifugation (Haemonetics, Braintree, MA). However, Cryptosporidium and Giardia (oo)cyst recoveries have varied greatly depending on the source water matrix and filters used1,14. A new tangential flow hollow-fiber ultrafiltration (HFUF) system has recently been shown to be more efficient and more robust at recovering Cryptosporidium oocysts and Giardia cysts from various water matrices; moreover, it is less expensive than other capsule filter options and can concentrate multiple pathogens simultaneously1-3,5-8,10,11. In addition, previous studies by Hill and colleagues demonstrated that the HFUF significantly improved Cryptosporidium oocysts recoveries when directly compared with the Envirochek HV filters4. Additional modifications to the current methods have also been reported to improve method performance. Replacing the acid dissociation procedure with heat dissociation was shown to be more effective at separating Cryptosporidium from the magnetic beads in some matrices9,13 .
This protocol describes a modified Method 1623 that uses the new HFUF filtration system with the heat dissociation step. The use of HFUF with this modified Method is a less expensive alternative to current EPA Method 1623 filtration options and provides more flexibility by allowing the concentration of multiple organisms.

A Modified EPA Method 1623 that Uses Tangential Flow Hollow-fiber Ultrafiltration and Heat Dissociation Steps to Detect Waterborne Cryptosporidium and Giardia spp.

This protocol describes the use of a tangential flow hollow-fiber ultrafiltration sample concentration system and a heat dissociation as alternative steps for the detection of waterborne Cryptosporidium and Giardia species using EPA Method 1623.

Eine Änderung, die EPA-Methode 1623 Tangential Flow Hohlfaser-Ultrafiltration und Wärme Dissoziationsstufen nutzt, um auf Wasserbasis Detect<em&gt; Cryptosporidium</em&gt; Und<em&gt; Giardia spp.</em

Cryptosporidium and Giardia species are two of the most prevalent protozoa that cause waterborne diarrheal disease outbreaks worldwide. To better ...

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 T cells in vitro with TCR- and co-stimulation in the presence of TGF&beta; and IL-2. This bears enormous potential for future therapies, however, the molecules and signaling pathways that control differentiation are largely unknown.
Primary T cells can be manipulated through ectopic gene expression, but common methods fail to target the most important naive state of the T cell prior to primary antigen recognition. Here, we provide a protocol to express ectopic genes in naive CD4 T cells in vitro before inducing Treg differentiation. It applies transduction with the replication-deficient adenovirus and explains its generation and production. The adenovirus can take up large inserts (up to 7 kb) and can be equipped with promoters to achieve high and transient overexpression in T cells. It effectively transduces naive mouse T cells if they express a transgenic Coxsackie adenovirus receptor (CAR). Importantly, after infection the T cells remain naive (CD44low, CD62Lhigh) and resting (CD25-, CD69-) and can be activated and differentiated into Tregs similar to non-infected cells. Thus, this method enables manipulation of CD4 T cell differentiation from its very beginning. It ensures that ectopic gene expression is already in place when early signaling events of the initial TCR stimulation induces cellular changes that eventually lead into Treg differentiation.

Adenoviral gene transfer into naive CD4 T cells with transgenic expression of the Coxsackie adenovirus receptor enables the molecular analysis of regulatory T cell differentiation in vitro.

Adenovirale Transduktion von Naive CD4 T-Zellen zu Treg Differenzierung Studieren

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 ...

Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological mechanisms involved during the disease is the T cell transfer model of colitis. In this model, immunodeficient mice (RAG-/- recipients) are reconstituted with naive CD4+ T cells from healthy wild type hosts.
This model allows examination of the earliest immunological events leading to disease and chronic inflammation, when the gut inflammation perpetuates but does not depend on a defined antigen. To study the potential role of antigen presenting cells (APCs) in the disease process, it is helpful to have an antigen-driven disease model, in which a defined commensal-derived antigen leads to colitis. An antigen driven-colitis model has hence been developed. In this model OT-II CD4+ T cells, that can recognize only specific epitopes in the OVA protein, are transferred into RAG-/- hosts challenged with CFP-OVA-expressing E. coli. This model allows the examination of interactions between APCs and T cells in the lamina propria.

In this antigen-driven colitis model, OT-II CD4+ T cells expressing a red fluorescent protein were adoptively transferred into RAG-/- mice that express a green fluorescent protein in mononuclear phagocytes (MPs). The hosts were challenged with Escherichia coli (E.coli) expressing the ovalbumin protein (OVA) fused to a cyan fluorescent protein (CFP).

Entwicklung einer Präsentation von Antigenen zu studieren Antigen-driven ulcerosa Modell durch Antigen-präsentierende Zellen zu T-Zellen präsentieren

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological ...

Precision-cut Mouse Lung Slices to Visualize Live Pulmonary Dendritic Cells

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for quantitative analysis of cell surface proteins on immune cells, but it provides no information on the localization and migration patterns of these cells within the lung. Similarly,&#160;chemotaxis assays can be performed to study the potential of cells to respond to chemotactic factors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these aforementioned techniques, the location of individual cell types within the lung can be readily visualized by generating Precision-cut Lung Slices (PCLS), staining them with commercially available, fluorescently tagged antibodies, and visualizing the sections by confocal microscopy. PCLS can be used for both live and fixed lung tissue, and the slices can encompass areas as large as a cross section of an entire lobe. We have used this protocol to successfully visualize the location of a wide variety of cell types in the lung, including distinct types of dendritic cells, macrophages, neutrophils, T cells and B cells, as well as structural cells such as lymphatic, endothelial, and epithelial cells. The ability to visualize cellular interactions, such as those between dendritic cells and T cells, in live, three-dimensional lung tissue, can reveal how cells move within the lung and interact with one another at steady state and during inflammation. Thus, when used in combination with other procedures, such as flow cytometry and quantitative PCR, PCLS can contribute to a comprehensive understanding of cellular events that underlie allergic and inflammatory diseases of the lung.

We describe a method for generating Precision-cut Lung Slices (PCLS) and immunostaining them to visualize the localization of various immune cell types in the lung. Our protocol can be extended to visualize the location and function of many different cell types under a variety of conditions.

Precision-cut Maus Lung Slices Live-Pulmonary Dendritische Zellen sichtbar zu machen

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for ...

An All-on-chip Method for Rapid Neutrophil Chemotaxis Analysis Directly from a Drop of Blood

Neutrophil migration and chemotaxis are critical for our body's immune system. Microfluidic devices are increasingly used for investigating neutrophil migration and chemotaxis owing to their advantages in real-time visualization, precise control of chemical concentration gradient generation, and reduced reagent and sample consumption. Recently, a growing effort has been made by the microfluidic researchers toward developing integrated and easily operated microfluidic chemotaxis analysis systems, directly from whole blood. In this direction, the first all-on-chip method was developed for integrating the magnetic negative purification of neutrophils and the chemotaxis assay from small blood volume samples. This new method permits a rapid sample-to-result neutrophil chemotaxis test in 25 min. In this paper, we provide detailed construction, operation and data analysis method for this all-on-chip chemotaxis assay with a discussion on troubleshooting strategies, limitations and future directions. Representative results of the neutrophil chemotaxis assay testing a defined chemoattractant, N-Formyl-Met-Leu-Phe (fMLP), and sputum from a chronic obstructive pulmonary disease (COPD) patient, using this all-on-chip method are shown. This method is applicable to many cell migration-related investigations and clinical applications.

This article provides the detailed method of performing a rapid neutrophil chemotaxis assay by integrating the on-chip neutrophil isolation from whole blood and the chemotaxis test on a single microfluidic chip.

Ein All-on-Chip-Verfahren zur schnellen Neutrophilen-Chemotaxis-Analyse direkt aus einem Blutstropfen

Neutrophil migration and chemotaxis are critical for our body's immune system. Microfluidic devices are increasingly used for investigating neutrophil ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

Überexpression und Reinigung des Menschen<em&gt; Cis</em&gt; -prenyltransferase in<em&gt; Escherichia coli</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

Ein neuartiges Feeder-freie System für die Massenproduktion von murinen Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&amp;#945;

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

Entwicklung und Validierung eines empfindsamen Einzelmolekül Array digitale Enzym-linked Immunosorbentprobe Assay für menschliche Interferon-α

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...