Diese Arbeit wurde durch ein Forschungsstipendium und ein Grant-in-Aid aus dem Food Nanotechnologie Projekt des Ministeriums für Landwirtschaft, Forsten und Fischerei Japans. Finanzierte

1. Perfusion der Leber <ol><li> Anesthetize ein erwachsenes Männchen Sprague-Dawley Ratten (bei 10 bis 15 Wochen alten, Körpergewicht 150-200 g) durch eine intraperitoneale Injektion von Natrium-Pentobarbital-Lösung (50 mg Natrium-Pentobarbital / kg Körpergewicht). </li><li> Legen Sie das Tier in eine rostfreie Pfanne und Spray 70% Ethanol auf seinen Bauch und Brustkorb. </li><li> Machen Sie einen kleinen Schnitt und Abziehen der Haut. Öffnen Sie den Unterleib durch den Schnitt mit einer Schere. </li><li> Bestätigen Sie den Speicherort der Pfortader, übergeben Sie einen chirurgischen Faden unter der Pfortader und lose Klumpen. </li><li> Clump den distalen Teil der Vena cava inferior und Vena mesenterica Blut Rückfluss in die Leber zu verhindern. Öffnen Sie die Brusthöhle, indem die Membran mit einer Schere, und setzen das Herz. </li><li> Legen Sie eine Kunststoff-Katheter (2 mm Durchmesser) in die Pfortader und ziehen Sie den Faden fest um den Katheter. </li><li> Verbinden Sie den Katheter zu einer Perfusion System, das mit Waschlösung (Ca 2 +-Mg 2 + free HBSS mit 0,5 mM EGTA, 10 mM HEPES und 4,2 mM NaHCO 3, pH 7,2) geladen wird vorgewärmt auf 37 ° C. </li><li> Begin Perfusion in situ mit einer Geschwindigkeit von 10 ml / min. Zur gleichen Zeit, einen Schnitt im rechten Vorhof Wand. Weiter Perfusion für 10 min. </li><li> Schalten Sie die Perfusionslösung zu Kollagenase-Lösung [10 mM HEPES HBSS und 4,2 NaHCO 3 mit Typ I Kollagenase ergänzt (0,05%) und Trypsin-Inhibitor (50 ug / ml); pH 7,5] und perfuse für 10 bis 20 min mit einer Rate von 10 ml / min, bis die Leber leicht anschwillt und zeigt das Zeichen für den Zerfall der Leber-Struktur unter Serosa. </li></ol> 2. Vorbereitung der Parenchymale Hepatocyte-rich Zellfraktion <ol><li> Entfernen und transferieren es vorsichtig in der Leber in ein steriles Becherglas mit 25 ml Kollagenase-Lösung. Mince in kleine Stücke schneiden mit der Schere. </li><li> Fügen Sie 75 ml kaltem MEM in die resultierende Zellsuspension. Nach einer sanften Pipettieren, Filtrat durch eine Zelle Sieb (100 um) zu entfernen, Bindegewebe und unverdaute Gewebeteile. </li><li> Das Filtrat in 50 ml konische Röhrchen und zentrifugieren bei 50 xg für 1 min bei 4 ° C (Bremse aus). </li><li> Den Überstand sorgfältig entfernen. Das Pellet mit kaltem MEM. </li><li> Wiederholen 2,4) dreimal. </li></ol> 3. Mixed Primary Kultur von Leberzellen <ol><li> Suspend die Zellen in 2,5 erhalten) in das Wachstumsmedium DMEM mit 10% Hitze-inaktiviertem FCS, mit 100 uM β-Mercaptoethanol, 10 pg / ml Insulin, 100 ug / ml Streptomycin und 100 U / ml Penicillin und Samen in Ergänzung zusammen fünf vor zehn Zellkulturflaschen (Fläche: 75 cm 2) bei einer Dichte von 6,7 x 10 4 Zellen / cm 2 (5 x 10 6 Zellen / Kolben / 12 -15 ml Medium). </li><li> Inkubieren Sie die flasksat 37 ° C in einer Atmosphäre von 5% CO 2 bis 95% Luft. </li><li> Ersetzen Sie das Nährmedium alle 2 bis 3 Tage Abständen. </li></ol> 4. Selektive Isolierung von Makrophagen <ol><li> Nach 12 Tagen der Kultur, vermehren Makrophagen auf der Zellschicht. Diese Zellen sind durch gegenseitige ausgesetzt Schütteln der Kulturflaschen bei 80 bis 120 Hüben pro Minute für 30 Minuten bei 37 ° C. </li><li> Übertragen Sie die Kulturmedium in 100 mm nicht Gewebekultur grade Plastikschalen. Pool des Mediums aus zwei T75-Kolben in eine Plastikschale. Refill Kulturflaschen mit dem Nährmedium und bringt sie in den Inkubator. </li><li> Inkubieren Plastikschalen für 30 min bei 37 ° C in einer Atmosphäre von 5% CO 2 bis 95% Luft. Makrophagen leicht auf nicht-Gewebekultur grade Plastikschalen unter dieser Inkubation Prozess anhängen, während andere kontaminierenden Zellen nicht. </li><li> Saugen Sie das Medium und sanft spülen Sie die Plastikschale mit PBS. Wiederholen Sie diesen Schritt dreimal. </li><li> 1 ml TrypLE Express-Lösung in die Schüssel, und Inkubation für 10min bei 37 ° C in einer Atmosphäre von 5% CO 2 bis 95% Luft. </li><li> Add 9 ml des Nährmediums. Vorsichtig abkratzen der beigefügten Makrophagen durch einen Zellschaber. </li><li> Übertragen Sie die Zellsuspension in ein konisches 15 ml Tube und zentrifugieren bei 180 xg für 5 min bei 25 ° C (Bremse). </li><li> Überstand verwerfen und 1 ml Wachstumsmedium. Distanzieren in einzelne Zellen durch kräftiges Pipettieren. Zählt die Anzahl der Zellen durch eine Zählkammer-Zählen. </li><li> Isolation Schritte können mit der gleichen Kulturflaschen auf zwei vor drei tägigen Intervallen für mehr als zwei Wochen wiederholt werden. </li><li> Isolierte Makrophagen in das Nährmedium in 3.1 beschrieben kultiviert werden. </li></ol> 5. Repräsentative Ergebnisse Ein Beispiel für eine gemischte primäre Kultur der erwachsenen Ratte Leberzellen ist in Abbildung 2 dargestellt. Parenchymale Hepatozyten verlieren die Epithelzellen Morphologie innerhalb von wenigen Tagen in Kultur, degenerieren oder Umwandlung in Fibroblasten-ähnlichen Zellen.Dann um den Tag von 6 bis 12, Phasenkontrast-helle, runde Makrophagen-ähnlichen Zellen vigourouly wuchern auf den Fibroblasten Zell-Blatt. Durch Schütteln der Kulturflaschen, sind Makrophagen leicht in das Kulturmedium suspendiert und nachfolgende Übertragung und Inkubation in Plastikschalen in selektiven Adhäsion von Makrophagen (Abbildung 3) führen. Die Makrophagen-ähnlichen Zellen können so früh wie Tag 8 geerntet werden, und die Zahlen erreicht maximal Werte an den Tagen 12 bis 14. Mehr als 10 6 Zellen können aus einer T75 Kulturflasche geerntet wiederholt werden auf 2 bis 3 Tage-Intervallen für mehr als zwei Wochen, so dass insgesamt Zelle Ertrag pro Flasche von 10 7 pro T75 Kulturflasche (Abbildung 3). Die Reinheiten der isolierten Makrophagen wurden 95 bis 99%, wie durch Durchflusszytometrie oder Immunzytochemie mit Ratten-Makrophagen-spezifischen Antikörpern, wie ED-1 (Abbildung 4), ED-3, OX-41 (Abbildung 4) und Iba1 9. Bewertet Die isolierten Zellen zeigen typische Merkmale von Makrophagen, wie aktive Phagozytose von polystylene Perlen (Abbildung 5), proliferative Reaktion auf rekombinantes GM-CSF, die Sekretion von entzündlichen / anti-inflammatorischen Zytokinen nach Stimulation mit LPS, und die Bildung von vielkernigen Riesenzellen 9. <img src="/files/ftp_upload/2757/2757fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Ein Schema für die selektive Isolierung und Aufreinigung von Makrophagen-ähnliche Zellen aus gemischten Primärkultur von Leberzellen. <img src="/files/ftp_upload/2757/2757fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Primäre Kultur der Leberzellen und die Proliferation von Makrophagen-ähnlichen Zellen. Der Pfeil zeigt eine mehrkernige Riesenzelle. Maßstab = 100 um. Nachdruck aus der Zeitschrift für Immunologische Methoden, Vol.360, 47-55 (2010) 9 mit Genehmigung von Elsevier. <img src="/files/ftp_upload/2757/2757fig3.jpg" alt="Abbildung 3" /> Abbildung 3. Selektive Isolierung von Makrophagen-ähnliche Zellen, die durch das Schütteln und Befestigungsart. Die Zellen wurden in das Kulturmedium durch Schütteln der Kolben suspendiert, anschließend in nicht-Gewebekultur grade Plastikschalen übertragen, und bei 37 ° C. Bereits 10 min nach dem Ausplattieren, Makrophagen-ähnlichen Zellen an das Gericht Oberfläche, während andere verunreinigende Fibroblasten-Zellen blieb suspendiert. Nach dem Spülen mit PBS wurde ein hochreines Makrophagen Bevölkerung erhalten. Diese Zellen gewonnen typischen Makrophagen Morphologie nach 40 min der Kultur, und mitotischen Zellen wurden häufig beobachtet (Pfeile). Nachträgliche Änderungen in Zellzahlen von Flaschen in verschiedenen Kultur Perioden erholt gezeigt. Die Werte sind im Durchschnitt ± SD 3-5 Flaschen. Die Experimente wurden dreimal wiederholt und die Daten sind durch unterschiedliche Symbole gekennzeichnet. Maßstab = 100 um. Nachdruck aus der Zeitschrift für Immunologische Methoden, Vol.360, 47-55 (2010) 9 mit Genehmigung von Elsevier. <img src="/files/ftp_upload/2757/2757fig4.jpg" alt="Abbildung 4" /> Abbildung 4. Immunzytochemische Färbung von Makrophagen-ähnliche Zellen mit monoklonalen Antikörpern gegen Ratten-Makrophagen. <img src="/files/ftp_upload/2757/2757fig5.jpg" alt="Abbildung 5" /> Abbildung 5. Phagozytose von FITC-markierten Mikrokugeln durch Makrophagen-ähnlichen Zellen.

<ol>
	<li>Crispe, I. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+liver+as+a+lymphoid+organ.">The liver as a lymphoid organ.</a> Annu. Rev. Immunol. 27, 147-163 (2009).</li><li>Roberts, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+Kupffer+cell+in+mediating+hepatic+toxicity+and+carcinogenesis.">Role of the Kupffer cell in mediating hepatic toxicity and carcinogenesis.</a> Toxicol. Sci. 96, 2-15 (2007).</li><li>Gao, B., Jeong, W. I., Tian, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver:+An+organ+with+predominant+innate+immunity.">Liver: An organ with predominant innate immunity.</a> Hepatology. 47, 729-736 (2008).</li><li>Janousek, J., Strmen, E., Gervais, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+murine+Kupffer+cells+by+centrifugal+elutriation.">Purification of murine Kupffer cells by centrifugal elutriation.</a> J. Immunol. Methods. 164, 109-117 (1993).</li><li>Olynyk, J. K., Clarke, 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=Isolation+and+primary+culture+of+rat+Kupffer+cells.">Isolation and primary culture of rat Kupffer cells.</a> J. Gastroenterol. Hepatol. 13, 842-845 (1998).</li><li>Heuff, G., Meyer, S., Beelen, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+rat+and+human+Kupffer+cells+by+a+modified+enzymatic+assay.">Isolation of rat and human Kupffer cells by a modified enzymatic assay.</a> J. Immunol. Methods. 174, 61-65 (1994).</li><li>Seglen, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+isolated+rat+liver+cells.">Preparation of isolated rat liver cells.</a> Methods Cell Biol. 13, 29-83 (1976).</li><li>Yoshioka, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+culture+and+expression+of+cytokine+mRNAs+by+lipopolysaccharide+in+bovine+Kupffer+cells.">Primary culture and expression of cytokine mRNAs by lipopolysaccharide in bovine Kupffer cells.</a> Vet. Immunol. Immunopathol. 58, 155-163 (1997).</li><li>Kitani, H., Takenouchi, T., Sato, M., Yoshioka, M., Yamanaka, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+isolation+method+for+macrophage-like+cells+from+mixed+primary+cultures+of+adult+rat+liver+cells.">A novel isolation method for macrophage-like cells from mixed primary cultures of adult rat liver cells.</a> J. Immunol. Methods. 360, 47-55 (2010).</li><li>Gorrell, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+fibrosis:+the+hepatocyte+revisited.">Liver fibrosis: the hepatocyte revisited.</a> Hepatology. 46, 1659-1661 (2007).</li></ol>

Alle Tiere wurden gehalten und betrieben in der Tierhaltung in der National Institute of Animal Health, nach den Richtlinien und Vorschriften her durch die Institution des Ausschusses für Tierversuche eingestellt.

Hier berichten wir über eine einfache und effiziente Methode, um Makrophagen aus dem gemischten primären Kulturen von adulten Ratten Leberzellen zu erhalten. Unsere Methode beruht zunächst auf dem Roman proliferative Aktivität von Makrophagen in der Mischkultur von Leberzellen, und anschließender Isolierung und Aufreinigung dieser Zellen auf der Basis ihrer biologischen Eigenschaften wie Makrophagen 9. Es kann möglich sein die Makrophagen in der gemischten Primärkultur von erwachsenen Leberzellen vermehrt könnte von Kupffer oder verwandten Zellen, die in den parenchymatösen Hepatozyten Bruchteil kontaminierten entstanden sein. Die Makrophagen könnte zu spezifischen Veränderungen der Umwelt durch Umwandlung von parenchymatösen Hepatozyten 10 und andere Fibroblasten-Zellen in der gemischten primären Kulturen verursacht geantwortet haben. Zusammenfassend bietet unsere Methode Isolierung von Makrophagen-ähnlichen Zellen in ausreichender Zahl und Reinheit aus dem primären Kultur von Rattenleber cellswithout mit hoch entwickelten Geräten und technischen Fähigkeiten. Darüber hinaus kann die Isolierung Verfahren mit der gleichen Kulturflasche für mehr als zwei Wochen wiederholt werden. Diese Methode könnte auch für andere Säugetierarten.

<table border="1"><tbody><tr><th> Name des Reagenzes </th><th> Firma </th><th> Katalog-Nummer </th><th> Kommentare (optional) </th></tr><tr><td> Natrium-Pentobarbital-Lösung </td><td> Kyoritsu Seiyaku </td><td> Somnopentyl Injection </td><td></td></tr><tr><td> Hanks balanced salt solution (HBSS) </td><td> Invitrogen </td><td> 14185-052 </td><td></td></tr><tr><td> Ethylenglykol tetraessigsäure (EGTA) </td><td> Sigma-Aldrich </td><td> E-4378 </td><td></td></tr><tr><td> Collagenase </td><td> Wako Pure Chemical Industries, Ltd </td><td> 032-10534 </td><td> Zelldissoziation grade (Lot Check erforderlich) </td></tr><tr><td> Trypsin-Inhibitor </td><td> Sigma-Aldrich </td><td> T-9128 </td><td></td></tr><tr><td> Eagle Minimum Essential Medium (MEM) </td><td> Sigma-Aldrich </td><td> M-4655 </td><td></td></tr><tr><td> Dulbecco modifiziertem Eagle-Medium (DMEM) </td><td> Sigma-Aldrich </td><td> D-6459 </td><td> Hohe Glukose-Typ </td></tr><tr><td> Fötalem Kälberserum (FCS) </td><td> HyClone </td><td> SH30070.03 </td><td> Wärme bei 56 ° C für 30 min inaktiviert. (Lot Check erforderlich) </td></tr><tr><td> TrypLE Express </td><td> Invitrogen </td><td> 12605 </td><td></td></tr><tr><td> Dulbeccos Phosphat-gepufferter Kochsalzlösung (PBS) </td><td> Invitrogen </td><td> 14190 </td><td> Ca 2 +-Mg 2 + frei </td></tr><tr><td> β-Mercaptoethanol </td><td> Sigma-Aldrich </td><td> M-7522 </td><td> Stock: 100 mM in destilliertem Wasser </td></tr><tr><td> Insulin </td><td> Sigma-Aldrich </td><td> I-5500 </td><td> Stock: 10 mg / ml in 0,1 N HCl </td></tr><tr><td> Penicillin / Streptomycin </td><td> Invitrogen </td><td> 15070 </td><td></td></tr><tr><td> Zellsieb </td><td> BD Biosciences </td><td> 352360 </td><td> Maschenweite: 100 pm </td></tr><tr><td> Zellkulturflasche </td><td> Sumitomo Bakelite Co., Ltd </td><td> MS-21250 </td><td> Fläche: 75 cm 2 </td></tr><tr><td> Non-Gewebekultur Plastikschalen </td><td> BD Biosciences </td><td> 351005 </td><td></td></tr><tr><td> Zellschaber </td><td> Corning Inc. </td><td> 3010 </td><td></td></tr><tr><td> Gegenseitige Schüttler </td><td> TAITEC </td><td> NR-1 </td><td></td></tr></tbody></table>

a simple and efficient method to isolate macrophages from mixed primary cultures of adult liver cells

Kupffer-Zellen sind Leber-spezifische Makrophagen und spielen eine wichtige Rolle in der physiologischen und pathologischen Funktionen der Leber 1-3. Obwohl die Isolierung Methoden der Leber Makrophagen wurden gut beschrieben 4-6, erfordern die meisten dieser Methoden gehobene Ausstattung, wie eine Zentrifugalkraft elutriator und technischen Fähigkeiten. Hier bieten wir eine neuartige Methode zur Leber Makrophagen in ausreichender Zahl und Reinheit zu erhalten aus gemischten primären Kulturen von adulten Ratten Leberzellen, wie in Abbildung 1 schematisch dargestellt. Nach Dissoziation der Leberzellen durch Zwei-Schritt-Perfusion-Methode 7,8, einem Bruchteil meist parenchymatösen Hepatozyten besteht vorbereitet und entkernt in T75 Zellkulturflaschen mit Kulturmedium DMEM und 10% FCS.Parenchymal Hepatozyten besteht verlieren die Epithelzellen Morphologie innerhalb von wenigen Tagen in Kultur, degenerieren oder Umwandlung in Fibroblasten-ähnlichen Zellen (Abbildung 2). Wie die Kultur geht, um den Tag 6, Phasenkontrast-hellen, starten rund Makrophagen-ähnlichen Zellen auf die Fibroblasten Zellrasen vermehren (Abbildung 2). Das Wachstum der Makrophagen-ähnlichen Zellen weiter und erreichen Höchstwerte um 12 Tage, für die Zellschicht auf der Flasche Oberfläche. Durch Schütteln der Kulturflaschen, sind Makrophagen leicht in das Kulturmedium suspendiert. Nachfolgende Übertragung und kurze Inkubation in Plastikschalen Ergebnis der selektiven Adhäsion von Makrophagen (Abbildung 3), wo andere kontaminierenden Zellen suspendiert bleiben. Nach mehreren Spülungen mit PBS, angeschlossen sind Makrophagen geerntet. Mehr als 10 6 Zellen können wieder aus dem gleichen T75 Zellkulturflasche auf zwei vor drei tägigen Intervallen geerntet werden für mehr als zwei Wochen (Abbildung 3). Die Reinheiten der isolierten Makrophagen wurden 95 bis 99%, wie mittels Durchflusszytometrie ausgewertet oder Immunzytochemie mit Ratten-Makrophagen-spezifischen Antikörpern (Abbildung 4). Die isolierten Zellen zeigen aktive Phagozytose von polystylene Perlen (Abbildung 5), proliferative Reaktion auf rekombinantes GM-CSF, die Sekretion von entzündlichen / anti-inflammatorischen Zytokinen nach Stimulation mit LPS und Bildung vielkernigen Riesenzellen 9. Abschließend stellen wir eine einfache und effiziente Methode, um Leber-Makrophagen in ausreichender Zahl und Reinheit zu erhalten, ohne komplexe Anlagen und skills.This Methode auf andere Säugetierarten können.

Watch this Scientific Journal Video about A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells at JoVE.com

A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

Eine neuartige Methode, um Makrophagen aus primären Kultur von Leberzellen zu erhalten, ist beschrieben. Diese Methode nutzt die Proliferation von Makrophagen in der Kultur, durch Schütteln der Kulturflaschen und Reinigung durch selektive Bindung an Plastikschalen gefolgt. Diese Technik bietet effiziente Leber Makrophagen ohne komplexe Anlagen und Fähigkeiten.

Eine einfache und effiziente Methode, um Makrophagen aus Mixed primären Kulturen von adulten Leber Zellen zu isolieren

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although ...

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Research

JoVE Journal

Immunology and Infection

37.9K Aufrufe.  National Institute of Agrobiological Sciences, Tsukuba, Japan. Eine neuartige Methode, um Makrophagen aus primären Kultur von Leberzellen zu erhalten, ist beschrieben. Diese Methode nutzt die Proliferation von Makrophagen in der Kultur, durch Schütteln der Kulturflaschen und Reinigung durch selektive Bindung an Plastikschalen gefolgt. Diese Technik bietet effiziente Leber Makrophagen ohne komplexe Anlagen und Fähigkeiten.

Serie: A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

Generation of Organotypic Raft Cultures from Primary Human Keratinocytes

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates epithelial differentiation. There are many uses for this system. For instance, the ability to grow three-dimensional organotypic raft cultures of keratinocytes has been an important milestone in the study of human papillomavirus (HPV)1. The life cycle of HPV is tightly linked to the differentiation of squamous epithelium2. Organotypic epithelial raft cultures as demonstrated here reproduce the entire papillomavirus life cycle, including virus production3,4,5. In addition, these raft cultures exhibit dysplastic lesions similar to those observed upon in vivo infection with HPV. Hence this system can also be used to study epithelial cell cancers, as well as the effect of drugs on epithelial cell differentiation in general. Originally developed by Asselineau and Prunieras6 and modified by Kopan et al.7, the organotypic epithelial raft culture system has matured into a general, relatively easy culture model, which involves the growth of cells on collagen plugs maintained at an air-liquid interface (Figure 1A). Over the course of 10-14 days, the cells stratify and differentiate, forming a full thickness epithelium that produces differentiation-specific cytokeratins. Harvested rafts can be examined histologically, as well as by standard molecular and biochemical techniques. In this article, we describe a method for the generation of raft cultures from primary human keratinocytes. The same technique can be used with established epithelial cell lines, and can easily be adapted for use with epithelial tissue from normal or diseased biopsies8. Many viruses target either the cutaneous or mucosal epithelium as part of their replicative life cycle. Over the past several years, the feasibility of using organotypic raft cultures as a method of studying virus-host cell interactions has been shown for several herpesviruses, as well as adenoviruses, parvoviruses, and poxviruses9. Organotypic raft cultures can thus be adapted to examine viral pathogenesis, and are the only means to test novel antiviral agents for those viruses that are not cultivable in permanent cell lines.

An in vitro method to mimic in vivo epithelial differentiation is described. Many viruses target epithelial cells as part of their viral life cycle, and this method provides a means of examining virus:host interactions that more closely resembles that which occurs in vivo. This technique can be used with primary keratinocytes, established cell lines, as well as normal or diseased biopsy tissue.

Generierung von Organotypische Floßkulturen aus primären humanen Keratinozyten

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates ...

Forschung

Immunologie und Infektion

Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue

Microglia account for approximately 12% of the total cellular population in the mammalian brain. While neurons and astrocytes are considered the major cell types of the nervous system, microglia play a significant role in normal brain physiology by monitoring tissue for debris and pathogens and maintaining homeostasis in the parenchyma via phagocytic activity 1,2. Microglia are activated during a number of injury and disease conditions, including neurodegenerative disease, traumatic brain injury, and nervous system infection 3. Under these activating conditions, microglia increase their phagocytic activity, undergo morpohological and proliferative change, and actively secrete reactive oxygen and nitrogen species, pro-inflammatory chemokines and cytokines, often activating a paracrine or autocrine loop 4-6. As these microglial responses contribute to disease pathogenesis in neurological conditions, research focused on microglia is warranted.
	Due to the cellular heterogeneity of the brain, it is technically difficult to obtain sufficient microglial sample material with high purity during in vivo experiments. Current research on the neuroprotective and neurotoxic functions of microglia require a routine technical method to consistently generate pure and healthy microglia with sufficient yield for study. We present, in text and video, a protocol to isolate pure primary microglia from mixed glia cultures for a variety of downstream applications. Briefly, this technique utilizes dissociated brain tissue from neonatal rat pups to produce mixed glial cell cultures. After the mixed glial cultures reach confluency, primary microglia are mechanically isolated from the culture by a brief duration of shaking. The microglia are then plated at high purity for experimental study.
	The principle and protocol of this methodology have been described in the literature 7,8. Additionally, alternate methodologies to isolate primary microglia are well described 9-12. Homogenized brain tissue may be separated by density gradient centrifugation to yield primary microglia 12. However, the centrifugation is of moderate length (45 min) and may cause cellular damage and activation, as well as, cause enriched microglia and other cellular populations. Another protocol has been utilized to isolate primary microglia in a variety of organisms by prolonged (16 hr) shaking while in culture 9-11. After shaking, the media supernatant is centrifuged to isolate microglia. This longer two-step isolation method may also perturb microglial function and activation. We chiefly utilize the following microglia isolation protocol in our laboratory for a number of reasons: (1) primary microglia simulate in vivo biology more faithfully than immortalized rodent microglia cell lines, (2) nominal mechanical disruption minimizes potential cellular dysfunction or activation, and (3) sufficient yield can be obtained without passage of the mixed glial cell cultures.
	It is important to note that this protocol uses brain tissue from neonatal rat pups to isolate microglia and that using older rats to isolate microglia can significantly impact the yield, activation status, and functional properties of isolated microglia. There is evidence that aging is linked with microglia dysfunction, increased neuroinflammation and neurodegenerative pathologies, so previous studies have used ex vivo adult microglia to better understand the role of microglia in neurodegenerative diseases where aging is important parameter. However, ex vivo microglia cannot be kept in culture for prolonged periods of time. Therefore, while this protocol extends the life of primary microglia in culture, it should be noted that the microglia behave differently from adult microglia and in vitro studies should be carefully considered when translated to an in vivo setting.

Isolating primary microglia from the cellular heterogeneity of the brain is essential to investigate their role in both physiological and pathological conditions. This protocol describes a mechanical isolation and mixed cell culture technique that provides high yield and high purity, viable primary microglial cells for in vitro study and downstream applications.

Primäre Mikroglia Isolation vom gemischten Zellkulturen von Gliazellen Neonatal Rattenhirngewebe

Microglia account for approximately 12% of the  total cellular population in the mammalian brain. While neurons and astrocytes are considered  the major ...

A Simple and Efficient Method to Detect Nuclear Factor Activation in Human Neutrophils by Flow Cytometry

Neutrophils are the most abundant leukocytes in peripheral blood. These cells are the first to appear at sites of inflammation and infection, thus becoming the first line of defense against invading microorganisms. Neutrophils possess important antimicrobial functions such as phagocytosis, release of lytic enzymes, and production of reactive oxygen species. In addition to these important defense functions, neutrophils perform other tasks in response to infection such as production of proinflammatory cytokines and inhibition of apoptosis. Cytokines recruit other leukocytes that help clear the infection, and inhibition of apoptosis allows the neutrophil to live longer at the site of infection. These functions are regulated at the level of transcription. However, because neutrophils are short-lived cells, the study of transcriptionally regulated responses in these cells cannot be performed with conventional reporter gene methods since there are no efficient techniques for neutrophil transfection. Here, we present a simple and efficient method that allows detection and quantification of nuclear factors in isolated and immunolabeled nuclei by flow cytometry. We describe techniques to isolate pure neutrophils from human peripheral blood, stimulate these cells with anti-receptor antibodies, isolate and immunolabel nuclei, and analyze nuclei by flow cytometry. The method has been successfully used to detect NF-&kappa;B and Elk-1 nuclear factors in nuclei from neutrophils and other cell types. Thus, this method represents an option for analyzing activation of transcription factors in isolated nuclei from a variety of cell types.

Neutrophils are the most abundant leukocytes in blood. Neutrophils possess transcriptionally regulated functions such as production of proinflammatory cytokines and inhibition of apoptosis. These functions can be studied with the method presented here, which allows detection and quantification of nuclear factors by flow cytometry in isolated nuclei

Eine einfache und effiziente Methode, um Nuclear Factor Aktivierung in menschlichen Neutrophilen Erkennen von Durchflusszytometrie

Neutrophils  are the most abundant leukocytes in peripheral blood. These cells are the first  to appear at sites of inflammation and infection, thus ...

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. Transfection with siRNA and plasmid DNA is an efficient tool for studying their function, but transfection of macrophages is not a trivial matter. Although many different approaches for transfection of eukaryotic cells are available, only few allow reliable and efficient transfection of macrophages, but reduced cell vitality and severely altered cell behavior like diminished capability for differentiation or polarization are frequently observed. Therefore a transfection protocol is required that is capable of transferring siRNA and plasmid DNA into macrophages without causing serious side-effects thus allowing the investigation of the effect of the siRNA or plasmid in the context of normal cell behavior. The protocol presented here provides a method for reliably and efficiently transfecting human THP-1 macrophages and monocytes with high cell vitality, high transfection efficiency, and minimal effects on cell behavior. This approach is based on Nucleofection and the protocol has been optimized to maintain maximum capability for cell activation after transfection. The protocol is adequate for adherent cells after detachment as well as cells in suspension, and can be used for small to medium sample numbers. Thus, the method presented is useful for investigating gene regulatory effects during macrophage differentiation and polarization. Apart from presenting results characterizing macrophages transfected according to this protocol in comparison to an alternative chemical method, the impact of cell culture medium selection after transfection on cell behavior is also discussed. The presented data indicate the importance of validating the selection for different experimental settings.

This protocol presents an efficient and reliable method to transfect human THP-1 macrophages with siRNA or plasmid DNA by electroporation with high transfection efficiency while maintaining high cell vitality and full macrophage capacity for differentiation and polarization.

Hocheffiziente Transfektion von humanen THP-1 Makrophagen durch Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. ...

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 ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

Eine einfache und effiziente Methode zu konstruieren Mutant Vacciniavirusvektoren

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.

Bone marrow cells cultured with granulocyte macrophage colony stimulating factor (GM-CSF) generate a heterogeneous culture containing macrophages and dendritic cells (DCs). This method highlights using MHCII and hyaluronan (HA) binding to differentiate macrophages from the DCs in the GM-CSF culture. Macrophages in this culture have many similarities to alveolar macrophages.

Erzeugung und Identifizierung von GM-CSF Abgeleitet Alveolare ähnlichen Makrophagen und dendritische Zellen aus Knochenmark der Maus

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. ...

Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the uremic milieu, oxidative stress, endotoxemia and the decreased clearance of circulating proinflammatory cytokines by producing a large number of acute-phase reactants. Experimental tools to study inflammation and the underlying role of hepatocytes are crucial to understand the regulation and contribution of hepatic cytokines to a systemic acute phase response and a prolonged pro-inflammatory scenario, especially in an intricate setting such as chronic kidney disease. Since studying complex mechanisms of inflammation in vivo remains challenging, resource-intensive and usually requires the usage of transgenic animals, primary isolated hepatocytes provide a robust tool to gain mechanistic insights into the hepatic acute-phase response. Since this in vitro technique features moderate costs, high reproducibility and common technical knowledge, primary isolated hepatocytes can also be easily used as a screening approach. Here, we describe an enzymatic-based method to isolate primary murine hepatocytes, and we describe the assessment of an inflammatory response in these cells using ELISA and quantitative real-time PCR.

Here, we show an enzymatic approach to isolate primary hepatocytes from adult mice, and we describe the quantification of an inflammatory response using ELISA and real-time PCR.

Die Induktion einer entzündlichen Reaktion in der Primär Hepatozyten-Kulturen von Mäusen

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the ...

A Non-invasive Way to Isolate and Phenotype Cells from the Conjunctiva

Traditionally, ocular surface cytology is studied with techniques such as spatula technology and brush technology. The problem with these techniques is that they may induce traumatic lesions on the surface of the eye, which can progress to scarring, eyelid deformity, limbal stem cell deficiency and in some cases, cause great discomfort to the subject. To avoid these clinical problems, impression cytology (IC) was developed to diagnose dry eye disease and later neoplasia, atopic disease, vernal keratoconjunctivitis and keratoconjunctivitis sicca. Typically, clinicians manually cut filter papers into required shapes and apply these to the ocular surface. Here, we describe how to perform IC using a commercially available medical device. This technique is explained here followed by immunophenotyping by flow cytometry. This technique requires less manual handling and causes less injury to the ocular surface.

The exposed normal ocular surface consists of cornea and conjunctiva. Epithelial cells, goblet cells and immune cells are present in the conjunctiva. Here, a non-invasive, technique of impression cytology is described using an impression cytology device and flow cytometry to analyze immune cells in the conjunctiva.

Ein nicht-invasiver Weg, um Zellen aus der Conjunctiva zu isolieren und zu phenotypen

Traditionally, ocular surface cytology is studied with techniques such as spatula technology and brush technology. The problem with these techniques is ...

An Efficient and Simple Method to Establish NK and T Cell Lines from Patients with Chronic Active Epstein-Barr Virus Infection

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed feeder cells, purified NK or T cells with as much as 10 mL of blood, or a high-dose of IL-2. This study presents a new method with a powerful and simple strategy to establish NK and T cell lines by culturing the peripheral blood mononuclear cells (PBMC) with the addition of recombinant human IL-2 (rhIL-2), and uses as little as 2 mL of whole blood. The cells can proliferate quickly in two weeks and be maintained for more than 3 months. With this method, 7 NK or T cell lines have been established with a high success rate. This method is simple, reliable, and applicable to establishing cell lines from more cases of CAEBV or NK/T cell lymphoma.

A simple method for obtaining NK and T cell clones from CAEBV patients was developed with high efficiency, a small amount of peripheral blood, and a low-dose of IL-2.

Eine effiziente und einfache Methode, NK und T-Zell-Linien von Patienten mit chronischen aktiven Epstein - Barr-Virus-Infektion zu etablieren

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed ...