JSO durch ein NIH Ruth L. Kirschstein NRSA (F32 DK091040) unterstützt wird, ist AK durch ein Postdoc-Stipendium von der American Diabetes Association (7-10-MI-05) unterstützt und wird durch einen AHH American Heart Association Gegründet Investigator Awards unterstützt (12EIA8270000). Durchflusszytometrie Experimente wurden in der VMC Durchflusszytometrie Shared Resource durchgeführt. Der VMC Durchflusszytometrie Shared Resource wird von der Vanderbilt Digestive Disease Research Center (DK058404) unterstützt.

1. Reagenzien und Zubehör Vor Beginn dieses experimentelle Protokoll, bereiten Sie die folgenden Reagenzien: <ol><li> 70% Ethanol </li><li> 1X PBS </li><li> 1X DPBS (ohne Ca und Mg) mit 0,5% BSA ergänzt </li><li> FACS-Puffer: 1X DPBS (ohne Ca und Mg), 2 mM EDTA und 1% FCS </li><li> ACK-Puffer: 150 mM NH 4 Cl, 10 mM KHCO 3 und 0,1 mM Na 2 EDTA in Wasser </li></ol> 2. Ernte und Herstellung von Fettgewebe <ol><li> Euthanize Mäusen nach IACUC genehmigten Modalitäten der einzelnen Institution. </li><li> Gründlich benetzen das Fell mit 70% Ethanol. </li><li> Einen Einschnitt auf der Ebene des Xiphoidbasis (unterer Teil des Brustbeins) und öffnen Sie die Brusthöhle, um das Herz aussetzen, kümmert sich nicht um irgendwelche großen Blutgefäße durchtrennen. </li></ol> Hinweis: An dieser Stelle ist es auch hilfreich, um die Membran intakt lassen so viel wiemöglich und eine Kerbe geschnitten aus der rechten Seite des Brustkorbs, um Blut zu ermöglichen und Perfusat zu fließen aus der Brusthöhle. <ol start="4"><li> Befestigen Sie den rechten Vorhof, um Blut zu ermöglichen und um das Perfusat Kreislaufsystem zu entkommen. </li></ol> Hinweis: Beim Umgang mit übergewichtigen Mäusen, überschüssige pericardial AT müssen entfernt werden, um Zugang zum Herzen zu ermöglichen. <ol start="5"><li> Fassen Sie das Herz mit einer Pinzette vorsichtig und legen Sie eine Nadel in die linke Herzkammer durch den Scheitelpunkt. Langsam versorgen die Maus mit 15 ml sterilem PBS. </li></ol> Hinweis: Reduzieren Sie die Anzahl der Perfusion, wenn die Lunge zu füllen und zu erweitern beginnen. <ol start="6"><li> Öffnen Sie die Bauchhöhle und entfernen Sie die perigonadal Fettpolster mit Sorgfalt, um alle gonadal Gewebe zu vermeiden. </li><li> Zeigen Fettpolster in ein Wiegeschiffchen auf Eis mit 2 ml 1X DPBS (ohne Mg oder Ca) mit 0,5% BSA ergänzt, und zerkleinern das AT in feine Stücke. </li></ol>Hinweis: Begrenzen Sie die Menge von AT pro wiegen Boot bis 1,2 g. Wenn die Menge von mehr als 1,2 g, teilen sie gleichmäßig zwischen zwei Booten wiegen. <ol start="8"><li> Halten AT Proben auf Eis und bereiten 3 ml Kollagenase Abbaulösung pro AT Probe, bestehend aus 1X DPBS mit 0,5% BSA, 10 mM CaCl 2 und 4 mg / ml Collagenase Typ II ergänzt. </li></ol> 3. Kollagenaseverdau <ol><li> Übertragen AT bis 50 ml konische Röhrchen, indem die Homogenat und Spülen das Boot mit 1 ml DPBS (0,5% BSA) und 3 ml Collagenase II Abbaulösung wiegen. </li><li> Inkubieren bei Homogenat in einer Rotationsrichtung Schüttler (200 Upm) bei 37 ° C für 20 min. </li><li> 10 ml DPBS (0,5% BSA) auf konische Röhrchen und auf Eis. </li><li> Man reibt Homogenat mehrmals mit einer serologischen Pipette 10 ml, und übergeben Zellsuspensionen durch 100 um-Filter in einer neuen 50 ml konischen Röhrchen. </li><li> Zentrifuge Zellsuspension bei 500 g für 10 min bei 4 &amp; dz. B., C. </li><li> Dekantieren Überstand und resuspendieren SVF Zellpellet in 3 ml ACK-Puffer zur Lyse verunreinigen Erythrozyten. </li><li> In 12 ml FACS-Puffer und Zentrifuge Zellsuspension bei 500 × g für 10 min bei 4 ° C. </li><li> Dekantieren Überstand und resuspendieren SVF Zellpellet in FACS-Puffer. </li></ol> Hinweis: Verwenden Sie die Größe des Zellpellets als Leitfaden für wie viel FACS Puffer resuspendieren in. Wenn das Zellpellet bedeckt den Boden des 50 ml konischen Röhrchen, verwenden 0,5-1 ml FACS-Puffer, andernfalls in 0,25-0,5 resuspendieren ml. <ol start="9"><li> Proben auf Eis stellen und bereiten 1:10 Verdünnung Aliquots jeder Probe zur Zellzählung durch Mischen von 40 ul FACS-Puffer, 50 ul Trypanblau-Lösung (0,2%), und 10 ul Zellsuspension. </li><li> Anzahl lebensfähiger Zellen basierend auf Trypanblauausschluß und verdünnte Zellsuspensionen zu einer Endkonzentration von 5-10 x 10 6 Zellen / ml. </li></ol> 4. Die Färbung der Zelloberflächenantigene <ol&gt; <li> In anti-Maus CD16/CD32 Antikörper (Fc-Block) auf eine Endkonzentration von 0,5-1 ug/10 6 Zellen und Inkubation auf Eis für 10 min. </li><li> Transfer-Proben (≥ 10 6 Zellen) bis 12 x 75 mm Polystyrol Rundbodengefäße. Bereiten getrennten Röhren, wenn die Analyse Geldautomaten und T-Zellen, und kombinieren Sie zusätzliche Zellen, um eine ausreichende Anzahl von Rohren vorzubereiten, um die erforderlichen Ausgleichs-und modifizierten Fluoreszenz minus eins (FMO) Kontrollen unterzubringen. </li></ol> Hinweis: Als ein Beispiel, bei der Quantifizierung des Anteils von Geldautomaten auf F4/80 und CD11b, die folgende Vergütung und FMO Kontrollen basieren, müssen vorbereitet werden: - Unstained (Zellen) - DAPI oder Propidiumiodid (PI) einzigen Fleck (Zellen, fügen Sie nicht Lebensfähigkeit Farbstoff bis Schritt 5.1) - F4/80 APC einzigen Fleck (Zellen oder Entschädigung Perlen) - CD11b FITC einzigen Fleck (Zellen oderEntschädigung Perlen) - FMO 1 (Zellen): Ratte IgG2a κ Isotypenkontrolle APC + CD11b FITC + Lebensfähigkeit Farbstoff (aufgenommen bei Schritt 5.1) - FMO 2 (Zellen): F4/80 APC + Ratte IgG2b κ Isotypenkontrolle FITC + Lebensfähigkeit Farbstoff (aufgenommen bei Schritt 5.1) <ol start="3"><li> In fluorophorkonjugierten Primärantikörper und / oder Isotypkontrollen in der entsprechenden Konzentration (siehe Tabelle von Reagenzien und Materialien). </li><li> Schützen Proben aus Licht und Inkubation bei 4 ° C für 30 min. </li><li> 2 ml FACS-Puffer und Zentrifuge Zellsuspension bei 500 xg für 5 Minuten bei 4 ° C. </li><li> Dekantieren Überstand und resuspendieren SVF Zellpellet in 2 ml FACS-Puffer. </li><li> Zentrifuge Zellsuspension 500 xg für 5 min bei 4 ° C und resuspendieren SVF Zellpellet in ≥ 400 ul FACS-Puffer. </li><li> Übertragen Proben bis 12 x 75 mm Polystyrol Rundbodengefäße mit einer 35 um Zellsieb tube tops ausgestattet.</li><li> Vor Licht und Aufbewahrung der Probe bei 4 ° C bis FACS-Analyse. </li></ol> Hinweis: Für optimale Ergebnisse sollte immeadiately Zellen analysiert werden, jedoch FACS-Analyse mit diesem Protokoll wurde erfolgreich auf markierten Zellen in FACS-Puffer gespeichert 1-2 h durchgeführt bei 4 ° C. Wenn Zellen, die an Lagerzeit zu erhöhen, dann können markierten Zellen mit 2% Paraformaldehyd bei 4 ° C für 24 Stunden vor der FACS-Analyse fixiert werden. Antikörper Unternehmen deuten darauf hin, dass für die markierten Zellen gespeichert werden können bis zu einer Woche, aber dies nicht im Rahmen dieses Verfahrens geprüft. 5. FACS-Analyse <ol><li> Vor FACS Analyse hinzuzufügen Lebensfähigkeit Farbstoff und entsprechende Kontrollen für Live / Dead Zelldiskriminierung ermöglichen. </li></ol> Hinweis: Zahlreiche Lebensfähigkeit Farbstoffe sind im Handel erhältlich, aber DAPI und PI empfohlen je nach Anregung / Emission Prof.iles der Fluorophor-konjugierten Antikörpern verwendet wird. DAPI und Propidiumiodid werden zu jeder Probe in einer Endkonzentration von 0,2 mg / ml zugegeben. <ol start="2"><li> Verwenden Sie einen ungefärbten negativen Kontrollprobe, vorzugsweise Zellen aus dem gleichen Gewebe wie die experimentellen Proben isoliert, um Seitwärtsstreuung (SSC) und Forward Scatter (FSC) so einstellen, dass die Zell-Population (n) von Interesse auf der Skala sind. </li></ol> Hinweis: Für die Analyse von AT SVF Zellen, es wird empfohlen, dass SSC in einer logarithmischen Skala gegen FSC in einer linearen Skala angezeigt werden. Die Verwendung einer logarithmischen Skala für SSC ist besonders wichtig bei der Analyse von ATMs, die oft sehr groß und Granulat. <ol start="3"><li> Zeichnen Sie eine erste Lichtstreuung Tor von der Art der Zelle (n), die analysiert basierend, und stellen Sie den Photomultiplier (PMT) gewinnen, so dass die ungefärbte Zellen auf der anderen von einem einzigen Parameter-Histogramm nach links (etwa auf 10 2 zentriert) sind für die entsprechenden Kanäle. &lt;/ Li&gt; </ol> Hinweis: Es wird empfohlen, Lymphozyten und Makrophagen (oder anderen myeloischen Zellen) gesondert aufgrund von Unterschieden in Autofluoreszenz analysiert werden. <ol start="4"><li> Benutzen gefärbten Kontrollen oder Antikörper-Einfang Entschädigung Perlen Multi-Color-Kompensation durchzuführen. </li></ol> Hinweis: Die Verwendung der Entschädigung Perlen wird empfohlen, jedoch Verträglichkeit jedes Antikörpers muss gewährleistet sein. Zum Beispiel kann eine Entschädigung Perlen nicht mit Kaninchen-Antikörper, in welchem ​​Fall isolierten Zellen erforderlich ist, um eine angemessene Kontrolle einzigen Fleck erhalten werden Kreuzreaktion. <ol start="5"><li> Set experimentellen Toren auf modifizierten FMO Kontrollen. </li><li> Stellen Sie die Durchflusszytometer, die entsprechende Anzahl von Ereignissen über die Prävalenz der Bevölkerung von Interesse basierend sammeln und aufzuzeichnen experimentellen Daten. </li><li> Export FCS-Dateien für die Offline-Analyse. Es gibt zahlreiche Programme für Durchfluss cytometry Datenanalyse. Wir empfehlen Cytobank, eine web-basierte Plattform, die investigatores zu speichern und zu analysieren und erzeugen Bilder von jedem Computer mit Internet-Zugang 18 erlaubt. Darüber hinaus bietet Cytobank die Fähigkeit, Daten öffentlich zugänglich sind oder der Zugriff auf Mitarbeiter. </li></ol>

<ol>
	<li>Rodbell, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolism+of+Isolated+Fat+Cells.+I.+Effects+of+Hormones+on+Glucose+Metabolism+and+Lipolysis.">Metabolism of Isolated Fat Cells. I. Effects of Hormones on Glucose Metabolism and Lipolysis.</a> The Journal of Biological Chemistry. 239, 375-380 (1964).</li><li>Danse, L. H., Verschuren, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fish+oil-induced+yellow+fat+disease+in+rats.+III.+Lipolysis+in+affected+adipose+tissue.">Fish oil-induced yellow fat disease in rats. III. Lipolysis in affected adipose tissue.</a> Veterinary Pathology. 15, 544-548 (1978).</li><li>Weisberg, S. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Obesity+is+associated+with+macrophage+accumulation+in+adipose+tissue.">Obesity is associated with macrophage accumulation in adipose tissue.</a> The Journal of Clinical Investigation. 112, 1796-1808 (2003).</li><li>Xu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+inflammation+in+fat+plays+a+crucial+role+in+the+development+of+obesity-related+insulin+resistance.">Chronic inflammation in fat plays a crucial role in the development of obesity-related insulin resistance.</a> The Journal of Clinical Investigation. 112, 1821-1830 (2003).</li><li>Mathis, D., Shoelson, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunometabolism:+an+emerging+frontier.">Immunometabolism: an emerging frontier.</a> Nature Reviews. Immunology. 11, 81 (2011).</li><li>Stefanovic-Racic, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cells+promote+macrophage+infiltration+and+comprise+a+substantial+proportion+of+obesity-associated+increases+in+CD11c++cells+in+adipose+tissue+and+liver.">Dendritic cells promote macrophage infiltration and comprise a substantial proportion of obesity-associated increases in CD11c+ cells in adipose tissue and liver.</a> Diabetes. 61, 2330-2339 (2012).</li><li>Liu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+deficiency+and+pharmacological+stabilization+of+mast+cells+reduce+diet-induced+obesity+and+diabetes+in+mice.">Genetic deficiency and pharmacological stabilization of mast cells reduce diet-induced obesity and diabetes in mice.</a> Nature Medicine. 15, 940-945 (2009).</li><li>Feuerer, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lean,+but+not+obese,+fat+is+enriched+for+a+unique+population+of+regulatory+T+cells+that+affect+metabolic+parameters.">Lean, but not obese, fat is enriched for a unique population of regulatory T cells that affect metabolic parameters.</a> Nature Medicine. 15, 930-939 (2009).</li><li>Nishimura, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD8++effector+T+cells+contribute+to+macrophage+recruitment+and+adipose+tissue+inflammation+in+obesity.">CD8+ effector T cells contribute to macrophage recruitment and adipose tissue inflammation in obesity.</a> Nature Medicine. 15, 914-920 (2009).</li><li>Winer, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normalization+of+obesity-associated+insulin+resistance+through+immunotherapy.">Normalization of obesity-associated insulin resistance through immunotherapy.</a> Nature Medicine. 15, 921-929 (2009).</li><li>Winer, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B+cells+promote+insulin+resistance+through+modulation+of+T+cells+and+production+of+pathogenic+IgG+antibodies.">B cells promote insulin resistance through modulation of T cells and production of pathogenic IgG antibodies.</a> Nature Medicine. 17, 610-617 (2011).</li><li>Wu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+invariant+natural+killer+T+cells+by+lipid+excess+promotes+tissue+inflammation,+insulin+resistance,+and+hepatic+steatosis+in+obese+mice.">Activation of invariant natural killer T cells by lipid excess promotes tissue inflammation, insulin resistance, and hepatic steatosis in obese mice.</a> Proceedings of the National Academy of Sciences of the United States of America. 109. , 1143-1152 (2012).</li><li>Wu, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eosinophils+sustain+adipose+alternatively+activated+macrophages+associated+with+glucose+homeostasis.">Eosinophils sustain adipose alternatively activated macrophages associated with glucose homeostasis.</a> Science. 332, 243-247 (2011).</li><li>Elgazar-Carmon, V., Rudich, A., Hadad, N., Levy, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophils+transiently+infiltrate+intra-abdominal+fat+early+in+the+course+of+high-fat+feeding.">Neutrophils transiently infiltrate intra-abdominal fat early in the course of high-fat feeding.</a> Journal of Lipid Research. 49, 1894-1903 (2008).</li><li>Talukdar, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophils+mediate+insulin+resistance+in+mice+fed+a+high-fat+diet+through+secreted+elastase.">Neutrophils mediate insulin resistance in mice fed a high-fat diet through secreted elastase.</a> Nature Medicine. 18, 1407-1412 (2012).</li><li>Hagman, D. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+and+quantifying+leukocyte+populations+in+human+adipose+tissue:+impact+of+enzymatic+tissue+processing.">Characterizing and quantifying leukocyte populations in human adipose tissue: impact of enzymatic tissue processing.</a> Journal of Immunological Methods. 386, 50-59 (2012).</li><li>Basu, S., Campbell, H. M., Dittel, B. N., Ray, A. <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+specific+cell+population+by+fluorescence+activated+cell+sorting+(FACS).">Purification of specific cell population by fluorescence activated cell sorting (FACS).</a> J. Vis. Exp. (41), e1546 (2010).</li><li>Kotecha, N., Krutzik, P. O., Irish, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Web-based+analysis+and+publication+of+flow+cytometry+experiments.">Web-based analysis and publication of flow cytometry experiments.</a> Current Protocols in Cytometry. Chapter 10, Unit 10 (2010).</li><li>Cho, C. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenic+role+of+LYVE-1-positive+macrophages+in+adipose+tissue.">Angiogenic role of LYVE-1-positive macrophages in adipose tissue.</a> Circulation Research. 100, e47-e57 (2007).</li><li>Roederer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral+compensation+for+flow+cytometry:+visualization+artifacts,+limitations,+and+caveats.">Spectral compensation for flow cytometry: visualization artifacts, limitations, and caveats.</a> Cytometry. 45, 194-205 (2001).</li></ol>

Wir haben nichts zu offenbaren.

Das zunehmende Interesse an der Rolle des Immunsystems in den metabolischen Konsequenzen der Adipositas hat die weit verbreitete Verwendung von Durchflusszytometrie, um Immunzellen des AT charakterisieren geführt. Obwohl die genaue Protokoll zwischen den Labors auf ihre eigene Erfahrung und die Ausstattung variieren, bezogen wird, sind die kritischen Schritte Kollagenaseverdau, differentielle Zentrifugation und Zelloberflächenantigen Kennzeichnung. Das Ziel des vorliegenden Artikels ist es, ein detailliertes Protokoll...

Historisch ist das Fettgewebe (AT) als inertes Organ Lipidspeicherung, die expandiert und kontrahiert in Reaktion auf Energiebilanz betrachtet. Wir verstehen jetzt, dass bei einem dynamischen endokrine Organ, das aktiv sondert eine Reihe von Hormonen, die direkten Einfluss auf das Fressverhalten und systemische Glukosehomöostase darstellt. Darüber hinaus in den letzten zehn Jahren hat es eine zunehmende Wertschätzung für die zahlreichen Populationen von Immunzellen mit Wohnsitz in der AT Stromazellen vaskulären Fraktion (SVF), sowie ihren Beitrag zur AT-Homöostase. Die Fähigkeit, die AT Adipozyten und SVF Verwendung einer Kollagenase-Verdau trennen gefolgt durch differentielle Zentrifugation wurde zuerst von Rodbell 1964 1 beschrieben. Collagenase II wird am häufigsten für Adipozyten und SVF Trennung aufgrund von Wartungsarbeiten von Adipozyten Insulin-Rezeptoren 1 verwendet. Schon früh, enzymatische Fraktionierung von AT wurde in erster Linie eingesetzt, um adipo studierenCyte Stoffwechsel und Präadipozyten zu isolieren. In jüngerer Zeit hat diese Technik, mit der weit verbreiteten Verfügbarkeit von Durchflusszytometern und der ständig steigenden Anzahl von kommerziell erhältlichen fluorophorkonjugierten Antikörper kombiniert, die Charakterisierung von AT Immunzellen erleichtert. Obwohl die Anwesenheit von Immunzellen im entzündeten AT hatte zuvor 2 beschrieben, die bahnbrechenden Arbeiten von Weisberg et al. und Xu et al. veröffentlicht im Jahr 2003 waren die ersten, die Anhäufung von AT Makrophagen (ATMs) in Fettleibigkeit, die inflammatorische Zytokine sezernieren und korrelieren mit AT-spezifische und systemische Insulinresistenz 3,4 dokumentieren. Diese Beobachtungen diente als Grundlage für ein neues Forschungsfeld vor kurzem geprägt, &quot;immunometabolism&quot;, 5 und wurden von Studien implizieren verschiedene Immunzellen Bevölkerungsgruppen, einschließlich dendritischen Zellen 6, 7 Mastzellen, T-Zellen 8 gefolgt -10, B-Zellen 11, 12 NKT-Zellen, Eosinophile 13 und Neutrophilen 14,15 in der Entwicklung von Adipositas Insulinresistenz. Das Ziel dieses Artikels ist es, eine detaillierte Beschreibung der Kollagenase Digest Technik verwendet, um Zellen des AT SVF zu isolieren und zu charakterisieren Geldautomaten und AT T-Zellen über Durchflusszytometrie bieten. Dieses Protokoll wurde für Maus optimiert wurde AT, aber die Zuschauer können von der Lektüre einen ausgezeichneten Artikel bieten umfangreiche Detail auf die Optimierung dieser Technik für den menschlichen AT 16 profitieren. Die Zielgruppe dieses Artikels enthält Ermittler mit begrenzter Erfahrung im Umgang mit Maus und AT Durchführung Durchflusszytometrie. Mehrere praktische Überlegungen zum Ausgleich zellulären Ertrag und Rentabilität mit Zeit und Ressourcen sowie eine optimale Durchflusszytometrie Kontrollen zur Charakterisierung AT Immunzellpopulationen vorgestellt. Zusätzlich zu unserem Protokoll werden die Leser referred einer aktuellen JoVE Artikel von Basu et al. für eine ausgezeichnete Diskussion über einige der technischen Aspekte der Durchflusszytometrie, um eine ordnungsgemäße Kontrollen und Kompensationen 17 umfassen.

<table border="1">
 <tbody>
 <tr>
 <td>Name</td>
 <td>Company</td>
 <td>Catalog #</td>
 <td></td>
 </tr>
 <tr>
 <td>DPBS (no Ca or Mg) 10 x 500 ml</td>
 <td>Life Technologies</td>
 <td>14190-250</td>
 <td></td>
 </tr>
 <tr>
 <td>DAPI</td>
 <td>Life Technologies</td>
 <td>D3571</td>
 <td></td>
 </tr>
 <tr>
 <td>BSA</td>
 <td>Sigma</td>
 <td>A2153-100G</td>
 <td></td>
 </tr>
 <tr>
 <td>collagenase</td>
 <td>Sigma</td>
 <td>C6885-5G</td>
 <td></td>
 </tr>
 <tr>
 <td>propidium iodide solution</td>
 <td>Sigma</td>
 <td>P4864-10 ml</td>
 <td></td>
 </tr>
 <tr>
 <td>stable stack 20 microliter</td>
 <td>Rainin</td>
 <td>SS-L10</td>
 <td></td>
 </tr>
 <tr>
 <td>20 microliter filter tips</td>
 <td>Rainin</td>
 <td>SRL10F</td>
 <td></td>
 </tr>
 <tr>
 <td>stable stack 250 microliter tips</td>
 <td>Rainin</td>
 <td>SS-L250</td>
 <td></td>
 </tr>
 <tr>
 <td>1000 microliter tips</td>
 <td>Rainin</td>
 <td>GPS-L1000</td>
 <td></td>
 </tr>
 <tr>
 <td>1000 microliter filter tips</td>
 <td>Rainin</td>
 <td>GP-L1000F</td>
 <td></td>
 </tr>
 <tr>
 <td>250 microliter Filter Tips</td>
 <td>Rainin</td>
 <td>SR-L200F</td>
 <td></td>
 </tr>
 <tr>
 <td>FC Block</td>
 <td>BD Biosciences</td>
 <td>553142</td>
 <td></td>
 </tr>
 <tr>
 <td>fisher 100mn strainers</td>
 <td>Fisherbrand</td>
 <td>22-363-549</td>
 <td></td>
 </tr>
 <tr>
 <td>medium weigh dish</td>
 <td>Fisherbrand</td>
 <td>02-202B</td>
 <td></td>
 </tr>
 <tr>
 <td>aluminum foil</td>
 <td>Fisherbrand</td>
 <td>1213100</td>
 <td></td>
 </tr>
 <tr>
 <td>mincing scissors</td>
 <td>Fisherbrand</td>
 <td>089531B</td>
 <td></td>
 </tr>
 <tr>
 <td>Vortex</td>
 <td>Fisherbrand</td>
 <td>2215365</td>
 <td></td>
 </tr>
 <tr>
 <td>50 ml conical tubes</td>
 <td>BD Falcon</td>
 <td>14-959-49A</td>
 <td></td>
 </tr>
 <tr>
 <td>filter top FACS tubes</td>
 <td>BD Falcon</td>
 <td>352235</td>
 <td></td>
 </tr>
 <tr>
 <td>10 ml pipette case 200</td>
 <td>BD Falcon</td>
 <td>1367520</td>
 <td></td>
 </tr>
 <tr>
 <td>round bottom tubes</td>
 <td>BD Falcon</td>
 <td>352058</td>
 <td></td>
 </tr>
 <tr>
 <td>5 ml syringe</td>
 <td>BD Falcon</td>
 <td>309646</td>
 <td></td>
 </tr>
 <tr>
 <td>V Bottom Plates</td>
 <td>Costar</td>
 <td>07-200-107</td>
 <td></td>
 </tr>
 <tr>
 <td>transfer bulb pipette</td>
 <td>Thermo Scientific</td>
 <td>13-711-22</td>
 <td></td>
 </tr>
 <tr>
 <td>Shaker</td>
 <td>Thermo Scientific</td>
 <td>11 676 071</td>
 <td></td>
 </tr>
 <tr>
 <td>Adhesive mat</td>
 <td>Thermo Scientific</td>
 <td>1368750</td>
 <td></td>
 </tr>
 <tr>
 <td>Cell Culture Centrifuge</td>
 <td>Sorvall</td>
 <td>75253839</td>
 <td></td>
 </tr>
 <tr>
 <td>Adapters</td>
 <td>Sorvall</td>
 <td>75003723</td>
 <td></td>
 </tr>
 <tr>
 <td>Rat anti-mouse CD16/CD32</td>
 <td>BD Biosciences</td>
 <td>553142</td>
 <td>Concentration: 0.5 - 1 &mu;g/ 106 cells</td>
 </tr>
 <tr>
 <td>Rat anti-mouse F4/80</td>
 <td>eBioscience</td>
 <td>17-4801</td>
 <td>Fluorophore conjugate: APC 
 Concentration: 0.2 &mu;g/ 106 cells 
 Isotype control catalog number: 17-4321</td>
 </tr>
 <tr>
 <td>Rat anti-mouse CD11b</td>
 <td>eBioscience</td>
 <td>11-0112</td>
 <td>Fluorophore conjugate: FITC 
 Concentration: 0.5 &mu;g/ 106 cells 
 Isotype control catalog number: 11/1/4031</td>
 </tr>
 <tr>
 <td>Armenian Hamster anti-mouse CD11c</td>
 <td>eBioscience</td>
 <td>12-0114</td>
 <td>Fluorophore conjugate: PE 
 Concentration: 0.8 &mu;g/ 106 cells 
 Isotype control catalog number: Dec-88</td>
 </tr>
 <tr>
 <td>Goat anti-mouse MGL1/2</td>
 <td>R&amp;D Systems</td>
 <td>FAB4297P</td>
 <td>Fluorophore conjugate: PE 
 Concentration: 0.1 &mu;g/ 106 cells 
 Isotype control catalog number: IC108P</td>
 </tr>
 <tr>
 <td>Goat anti-mouse CD206</td>
 <td>R&amp;D Systems</td>
 <td>FAB2535P</td>
 <td>Fluorophore conjugate: PE 
 Concentration: 0.1 &mu;g/ 106 cells 
 Isotype control catalog number: IC108P</td>
 </tr>
 <tr>
 <td>Rat anti-mouse CCR2</td>
 <td>R&amp;D Systems</td>
 <td>FAB5538P</td>
 <td>Fluorophore conjugate: PE 
 Concentration: 0.1 &mu;g/ 106 cells 
 Isotype control catalog number: IC013P</td>
 </tr>
 <tr>
 <td>Armenian Hamster anti-mouse TCR&beta;</td>
 <td>BD Biosciences</td>
 <td>553174</td>
 <td>Fluorophore conjugate: APC 
 Concentration: 0.2 &mu;g/ 106 cells 
 Isotype control catalog number: 553956</td>
 </tr>
 <tr>
 <td>Rat anti-mouse CD8a</td>
 <td>BD Biosciences</td>
 <td>552877</td>
 <td>Fluorophore conjugate: PE-Cy7 
 Concentration: 0.8 &mu;g/ 106 cells 
 Isotype control catalog number: 552784</td>
 </tr>
 <tr>
 <td>Rat anti-mouse CD4</td>
 <td>BD Biosciences</td>
 <td>557956</td>
 <td>Fluorophore conjugate: Alexa Fluor 700 
 Concentration: 0.2 &mu;g/ 106 cells 
 Isotype control catalog number: 557963</td>
 </tr>
 </tbody>
</table>
Table 1. List of Materials and Reagents.

isolation of adipose tissue immune cells

Die Entdeckung erhöht Makrophageninfiltration im Fettgewebe (AT) von übergewichtigen Nagern und Menschen hat zu einer Intensivierung des Interesses an Immunzellen Beitrag zur lokalen und systemischen Insulinresistenz geführt. Isolierung und Quantifizierung der verschiedenen Immunzellpopulationen in schlanken und adipösen AT ist heute ein häufig genutzt Technik in immunometabolism Laboratorien; noch extremer Sorgfalt muss sowohl in Stromazellen vascular cell Isolation und in der Durchflusszytometrie-Analyse gemacht werden, so dass die erhaltenen Daten zuverlässig und interpretierbar ist . In diesem Video zeigen wir, wie man Hackfleisch, verdauen, und zu isolieren, das Immunsystem Zellen angereicherte Stromazellen vaskulären Fraktion. Anschließend zeigen wir, wie man Antikörper Label Makrophagen und T-Lymphozyten und wie man richtig Tor auf sie in der Durchflusszytometrie Experimente. Repräsentative Durchflusszytometrie Plots aus fettarm-fed schlank und fettreichen-fed fettleibigen Mäusen vorgesehen sind. Ein wichtiges Element dieser Analyse ist die Verwendung von Antikörpern, die zu tunfluoresziert nicht in Kanälen, in denen AT Makrophagen sind natürlich autofluoreszierenden, sowie die Verwendung der richtigen Kompensation Kontrollen.

Collagenase Verdauung von AT durch differentielle Zentrifugation wurde die SVF von Nebenhoden Fettpolster männlichen C57BL/6J-Mäusen isolieren zugeführt einer fettarmen (10% kcal aus Fett) oder hohen Fett (60% kcal aus Fett) Ernährung (LFD und HFD , jeweils) für 16 Wochen. Zellen des SVF wurden dann mit einem Fluorophor konjugiert primären Antikörpern markiert, um den Anteil von lebensfähigen Geldautomaten (1) und T-Zellen (2) über FACS-Analyse quantifiziert. Initial Gating, ei...

Watch this Scientific Journal Video about Isolation of Adipose Tissue Immune Cells at JoVE.com

Isolation of Adipose Tissue Immune Cells

Fettgewebe (AT) ist ein Ort der intensiven Immunzellaktivierung und Interaktion. Fast alle Zellen des Immunsystems sind in AT und ihre Verhältnisse werden durch Fettleibigkeit verändert. Proper Isolierung, Quantifizierung und Charakterisierung von AT Immunzellpopulationen sind entscheidend für das Verständnis ihrer Rolle in immunometabolic Krankheit.

Isolation von Fettgewebe Immune Cells

The discovery of increased macrophage infiltration in  the adipose tissue (AT) of obese rodents and humans has led to an  intensification of interest in ...

Research

JoVE Journal

Immunology and Infection

35.9K Aufrufe.  Vanderbilt University School of Medicine. Fettgewebe (AT) ist ein Ort der intensiven Immunzellaktivierung und Interaktion. Fast alle Zellen des Immunsystems sind in AT und ihre Verhältnisse werden durch Fettleibigkeit verändert. Proper Isolierung, Quantifizierung und Charakterisierung von AT Immunzellpopulationen sind entscheidend für das Verständnis ihrer Rolle in immunometabolic Krankheit.

Serie: Isolation of Adipose Tissue Immune Cells

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

Mess-Calpain Aktivität in fixierten und lebenden Zellen mittels Durchflusszytometrie

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

Forschung

Immunologie und Infektion

Quantitative Assessment of Immune Cells in the Injured Spinal Cord Tissue by Flow Cytometry:  a Novel Use for a Cell Purification Method

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true quantitative analysis of cellular inflammation. Flow cytometry is a quick alternative method to quantify immune cells in the injured brain or spinal cord tissue. Historically, flow cytometry has been used to quantify immune cells collected from blood or dissociated spleen or thymus, and only a few studies have attempted to quantify immune cells in the injured spinal cord by flow cytometry using fresh dissociated cord tissue. However, the dissociated spinal cord tissue is concentrated with myelin debris that can be mistaken for cells and reduce cell count reliability obtained by the flow cytometer. We have advanced a cell preparation method using the OptiPrep gradient system to effectively separate lipid/myelin debris from cells, providing sensitive and reliable quantifications of cellular inflammation in the injured spinal cord by flow cytometry. As described in our recent study (Beck &amp; Nguyen et al., Brain. 2010 Feb; 133 (Pt 2): 433-47), the OptiPrep cell preparation had increased sensitivity to detect cellular inflammation in the injured spinal cord, with counts of specific cell types correlating with injury severity. Critically, novel usage of this method provided the first characterization of acute and chronic cellular inflammation after SCI to include a complete time course for polymorphonuclear leukocytes (PMNs, neutrophils), macrophages/microglia, and T-cells over a period ranging from 2 hours to 180 days post-injury (dpi), identifying a surprising novel second phase of cellular inflammation. Thorough characterization of cellular inflammation using this method may provide a better understanding of neuroinflammation in the injured CNS, and reveal an important multiphasic component of neuroinflammation that may be critical for the design and implementation of rational therapeutic treatment strategies, including both cell-based and pharmacological interventions for SCI.

Quantification of cellular inflammation in the injured/pathological CNS by flow cytometry is complicated by lipid/myelin debris that can have similar size and granulation to cells, decreasing sensitivity/accuracy. We have advanced a cell preparation method to remove myelin debris and improve cell detection by flow cytometry in the injured spinal cord.

Quantitative Beurteilung der Immunzellen in das verletzte Rückenmark Tissue mittels Durchflusszytometrie: eine neue Verwendung für eine Zelle Reinigungsverfahren

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true ...

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

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 ...

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

Durchflusszytometrie-Analyse von Immunzellen innerhalb Murine Aorten

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

Isolation of Functional Cardiac Immune Cells

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5 These immune cells, which include mast cells, T-cells and macrophages; store and release a variety of biologically active mediators including cytokines and proteases such as tryptase.6-8 These mediators have been shown to be key players in extracellular matrix metabolism by activating matrix metalloproteinases or causing collagen accumulation by modulating the cardiac fibroblasts' function.9-11 However, available techniques for isolating cardiac immune cells have been problematic because they use bacterial collagenase to digest the myocardial tissue. This technique causes activation of the immune cells and thus a loss of function. For example, cardiac mast cells become significantly less responsive to compounds that cause degranulation.12 Therefore, we developed a technique that allows for the isolation of functional cardiac immune cells which would lead to a better understanding of the role of these cells in cardiac disease.13, 14
This method requires a familiarity with the anatomical location of the rat's xiphoid process, axilla and falciform ligament, and pericardium of the heart. These landmarks are important to increase success of the procedure and to ensure a higher yield of cardiac immune cells. These isolated cardiac immune cells can then be used for characterization of functionality, phenotype, maturity, and co-culture experiments with other cardiac cells to gain a better understanding of their interactions.

This method for isolating functional immune cells from the heart provides an alternative to the conventional methods of collagenase digestion, which causes unwanted immune cell activation, resulting in a decreased responsiveness of these cells. Our method of isolation yields functional cardiac immune cells by avoiding problems associated with enzymatic digestion.

Isolierung von Functional Cardiac Immune Cells

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5  These immune cells, which ...

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

Durchflusszytometrische Isolation of Primary Murine Type II Alveolarepithelzellen für Funktionelle und Molekulare Studien

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

Isolation und Analyse über Durchflusszytometrie von Immunzellen aus dem ischämischen Gehirn der Maus

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident immune cell populations in the oral mucosa and gingiva. We have established a technique for the isolation and study of immune cells from murine gingival tissues, an area of constant microbial exposure and a vulnerable site to a common inflammatory disease, periodontitis. Our protocol allows for a detailed phenotypic characterization of the immune cell populations resident in the gingiva, even at steady state. Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network should aid towards investigating the mechanisms involved in oral immunity and periodontal homeostasis, but will also advance our understanding of the mechanisms involved in local immunopathology.

We have established a technique for the isolation, phenotypic characterization and functional analysis of immune cells from murine gingiva.

Isolierung, Charakterisierung und funktionelle Untersuchung des Zahnfleisch Immune Cell Network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...

Reliable and High Efficiency Extraction of Kidney Immune Cells

Immune system activation occurs in multiple kidney diseases and pathophysiological processes. The immune system consists of both adaptive and innate components and multiple cell types. Sometimes, the cell type of interest is present in very low numbers among the large numbers of total cells isolated from the kidney. Hence, reliable and efficient isolation of kidney mononuclear cell populations is important in order to study the immunological problems associated with kidney diseases. Traditionally, tissue isolation of kidney mononuclear cells have been performed via enzymatic digestions using different varieties and strengths of collagenases/DNAses yielding varying numbers of viable immune cells. Recently, with the development of the mechanical tissue disruptors for single cell isolation, the collagenase digestion step is avoided and replaced by a simple mechanical disruption of the kidneys after extraction from the mouse. Herein, we demonstrate a simple yet efficient method for the isolation of kidney mononuclear cells for every day immune cell extractions. We further demonstrate an example of subset analysis of immune cells in the kidney. Importantly, this technique can be adapted to other soft and non-fibrous tissues such as the liver and brain.

Techniques that are reliable and efficient for the isolation of kidney immune cells are needed for downstream applications. This requires surface antibody labeling of a small number of kidney immune cells. Herein, we describe a concise method for isolation of kidney immune cells that seemingly achieves this goal.

Zuverlässig und High Efficiency Extraktion von Nieren-Immunzellen

Immune system activation occurs in multiple kidney diseases and pathophysiological processes.  The immune system consists of both adaptive and innate ...

Isolation, Expansion, and Adipogenic Induction of CD34+CD31+ Endothelial Cells from Human Omental and Subcutaneous Adipose Tissue

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor response to insulin, local hypoxia, and inflammation. By stimulating the differentiation of functional white adipocytes from progenitors, radical hypertrophy of the adipocyte population can be prevented and, consequently, the metabolic health of adipose tissue can be improved along with a reduction of inflammation. Also, by stimulating a differentiation of beige/brown adipocytes, the total body energy expenditure can be increased, resulting in weight loss. This approach could prevent the development of obesity co-morbidities such as type 2 diabetes and cardiovascular disease.
This paper describes the isolation, expansion, and differentiation of white and beige adipocytes from a subset of human adipose tissue endothelial cells that co-express the CD31 and CD34 markers. The method is relatively cheap and is not labor-intensive. It requires access to human adipose tissue and the subcutaneous depot is suitable for sampling. For this protocol, fresh adipose tissue samples from morbidly obese subjects [body mass index (BMI) &#62;35] are collected during bariatric surgery procedures. Using a sequential immunoseparation from the stromal vascular fraction, enough cells are produced from as little as 2&#8211;3 g of fat. These cells can be expanded in culture over 10&#8211;14 days, can be cryopreserved, and retain their adipogenic properties with passaging up to passage 5&#8211;6. The cells are treated for 14 days with an adipogenic cocktail using a combination of human insulin and the PPAR&#947; agonist-rosiglitazone.
This methodology can be used for obtaining proof of concept experiments on molecular mechanisms that drive adipogenic responses in adipose endothelial cells, or for screening new drugs that can enhance the adipogenic response directed either towards white or beige/brown adipocyte differentiation. Using small subcutaneous biopsies, this methodology can be used to screen out non-responder subjects for clinical trials aimed to stimulate beige/brown and white adipocytes for the treatment of obesity and co-morbidities.

The differentiation of white and beige adipocytes from adipose tissue vascular progenitors bears potential for metabolic improvement in obesity. We describe protocols for a CD34+CD31+ endothelial cell isolation from human fat and for a subsequent in vitro expansion and differentiation into white and beige adipocytes. Several downstream applications are discussed.

Isolation, Expansion und Adipogenen Induktion von CD34 + CD31 + Endothelial Zellen aus menschlichen Omentalis und subkutanem Fettgewebe

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor ...