Diese Arbeit wurde durch ein UNCF-Merck Graduate Science Research Dissertation Fellowship (JLM), National Heart, Lung, and Blood Institute gewährt RO1-HL-62228 (JSJ) und RO1-HL-073990 (JSJ) unterstützt.

1. Vorbereitung Hanks &#39;Balance Salt Solution (HBSS) <ol><li> Autoclave 1 L Becherglas, absolvierte 1000 ml Zylinder und Rührstab. </li><li> Bereiten Sie die HBSS Pulver Lösung im Autoklaven 1 L Becherglas auf die 900 mL Marke mit doppelt destilliertem Wasser. </li><li> Mix HBSS-Lösung mit 3,4 g Hepes Na + Salz-und pH-Wert auf einen Wert zwischen 7,35-7,41 entweder mit konzentrierter HCL oder NaOH. </li><li> Sobald der gewünschte pH erreicht ist und bleibt stabil, 5 g Rinderserumalbumin, um die Lösung bei Raumtemperatur, bis die Albumin vollständig gelöst ist. </li><li> Fügen Sie 10 ml des Antibiotikums, antimyotic, um die Lösung und doppelt destilliertem Wasser, bis das Gesamtvolumen auf 1000 mL-Marke. </li><li> Rühren Sie die Lösung gründlich. </li><li> Filtern Sie die HBSS-Lösung und teilen ihn in 50 mL Falcon-Röhrchen für die spätere Verwendung. Weitere Rohre können eingefroren und verwendet werden zu einem späteren Zeitpunkt. </li></ol> 2. Tierische VORBEREITUn - Einfügen von Trachealtubus <ol><li> Bereiten Sie ein Tablett mit den erforderlichen Instrumenten und Materialien für das Einführen eines Trachealtubus (dh Zangen, Scheren, Trachealtubus, 3-0 schwarz, geflochten, Kleintier-Lüfter, etc.). </li><li> Sobald die Ratte ist völlig betäubt mit einer Ketamin / Xylazin-Cocktail in das Verhältnis von 2 zu 1 Ketamin Xylazin, rasieren den Bauch, Brust und Hals der Ratte. Um sicherzustellen, dass eine tiefe Narkose Zustand erreicht worden ist, eine Prise der Ratte Zehen-und Scheck-Pedal Rückzug der Extremität. Achten Sie darauf, die Ratte ist tief narkotisiert, bevor alle Schnitte. </li><li> Legen Sie die Ratte mit dem Kopf auf ein Schneidebrett geäußert, dass bei einem Winkel von 30 ° geneigt ist </li><li> Machen Sie einen Hautschnitt von der Mitte des Bauches unterhalb des Unterkiefers. Dissect entfernt die Muskeln der vorderen Dreieck des Halses an der Luftröhre aussetzen. </li><li> Legen Sie einen 3:0-schwarz geflochten und unter der Luftröhre. </li><li> Machen Sie einen Schnitt in die Luftröhre zwischen den knorpeligen Ringe eined Legen Sie die Trachealtubus. Binden Sie die Naht fest mit dem Trachealtubus in die Luftröhre zu sichern. </li><li> Verbinden Sie den Trachealtubus zu einem kleinen Tier Ventilator. </li></ol> 3. Instrumenten-Aufbereitung für Immunzellen Isolation <ol><li> Bereiten Sie ein Tablett mit den Instrumenten für die Immunzellen isoliert Verfahren (dh Zangen, Scheren, HBSS, beschnitten weiche Teflon 24 Gauge Baxter intravasalen über die Nadel schnell cath) </li><li> Bereiten Sie zwei 10cc Spritzen, füllen Sie eine Spritze mit HBSS für die Einführung der Puffer in den Herzbeutel und behalten uns das andere für das Absaugen des HBSS enthaltenden Zellen. 3.2.1. Reduzieren Sie die Teflon Spitze des 24 Gauge Baxter intravasalen über die Nadel schnell cath (1,6 cm) mit 1 / 2 bis 3 / 4 seiner ursprünglichen Länge für die Isolierung Verfahren. </li><li> Legen Sie alle Spritzen, Puffer und eine leere 15-ml-Falcon Tube (die extrahierten Flüssigkeit halten, sobald Vorgang abgeschlossen ist) auf Eis. </li></ol> &lt;p class = &quot;jove_title&quot;&gt; 4. Immunzellen Isolation <ol><li> Machen Sie einen Medianschnitt in der Bauchwand (entlang der Linea alba), bis Sie die Xiphoidbasis erreichen. </li><li> Ab leicht seitlich an den Xiphoidbasis machen bilateralen Einschnitte durch den Brustkorb weiterhin in einem Winkel in Richtung Achselhöhle. Achten Sie darauf, nicht in die Lunge, Herz oder Perikard perforieren. </li><li> Nachdem die Schnitte auf beiden Seiten des Brustbeins abgeschlossen sind, sezieren entfernt die Membran von der unteren Rippen, Schneiden lateral nach medial. Hinweis: Seien Sie immer wissen, wo der Herzbeutel eine Verbindung mit dem Zwerchfell und Brustbein, um Schäden zu vermeiden. Verwenden Sie das Lig. falciforme als Wahrzeichen zu beachten, wo der Herzbeutel auf der gegenüberliegenden Seite der Membran angebracht ist. Auch, wenn das Perikard geschnitten wird, ist die Fähigkeit, Zellen zu isolieren verloren. </li><li> Heben Sie die eine Seite des Brustkorbs mit der Brusthöhle und dem Herzen (immer noch von den Herzbeutel gekapselt) aussetzen.</li><li> Nehmen Sie die HBSS gefüllte Spritze und finden Sie einen Punkt in den Herzbeutel etwa auf halbem Weg zwischen dem Brustbein und dem Herzen (vorzugsweise mehr rostral als caudal) und legen Sie die Teflon Spitze des verkürzten schnell cath durch beide Schichten des Herzbeutels. </li><li> Sobald der Katheter an Stelle vorsichtig vorgehen füllen den Herzbeutel Raum mit 2 bis 3 ml HBSS-Puffer. Sie werden sofort wissen, ob der Katheter richtig platziert ist, wenn man den Herzbeutel Raum erweitert kündigen. Wenn keine Füllung ist Zeuge, zurückzutreten und die Position des Quick-kath. </li><li> Wenn der Herzbeutel gefüllt ist, nehmen die anderen 10 ml-Spritze und setzen Sie die Spitze wieder in den Herzbeutel und beginnen Absaugen des Puffers. Der gleiche Eintrag Loch verwendet werden kann oder eine andere geschaffen werden kann. Sicherstellen, dass die Spitze nicht zu nahe kommen, um die Wände der Herzbeutel und gleichzeitig abgesaugt, besonders an der Unterseite des Herzens. Dies könnte zu Punktion des Herzbeutels und Verlust von Zellen enthaltende Zelle führens. </li><li> Während das Absaugen des Puffers, kann die Katheterspitze um in den Herzbeutel bewegt werden, um so viel Puffer wie möglich zu sammeln. </li></ol> Hinweis: Wenn dabei reißt ihr leicht den Herzbeutel und das Loch an der Spitze des Herzens größer als die ursprüngliche Loch, keine Panik. Solange der Herzbeutel noch umgibt genug des Herzens kann man noch durchführen anderen waschen über das Herz, wenn auch mit einer geringeren Menge an Puffer. Bei der Erhebung einer optimalen Menge an Zellen, sind drei Minuten vor vier zusätzliche Waschgänge nötig. <ol start="9"><li> Legen Sie die extrahierten Puffer in den Falcon-Röhrchen auf Eis gelegt wurden. </li><li> Nehmen Sie Herz, Lunge und / oder andere Gewebe, wenn für weitere Studien erforderlich. Nach der Sammlung von Immunzellen und anderen Geweben, muss die Ratte schnell und human eingeschläfert werden. </li><li> Centrifuge die gesammelten Immunzellen Proben für 10 min bei 200 g bei 4 ° C. Halten Sie das Röhrchen auf Eiswenn nicht in Gebrauch. </li><li> Entfernen Sie den Überstand und rekonstruieren das Pellet in 1 ml Hyclone Buffer oder HBSS. </li></ol> Hinweis: Es ist wichtig, um die Anzahl der Immunzellen aus jedem Herzen nach der Immunzellen isoliert Verfahren erhalten zählen. 5. Repräsentative Ergebnisse: Ein Beispiel von isolierten kardialen Mastzellen mit dieser Technik und gefärbt mit Toluidinblau und Safranin O / Alcianblau erzielten Ergebnisse sind in Abbildung 2a und 2b dargestellt, jeweils. Wie in Abbildung 3, die Herz-Mast zu sehen ist, behielten ihre Fähigkeit, Histamin nach der Behandlung mit bekannten Aktivatoren wie Verbindung 48/80 und Substanz P als in Morgan et al Release. 9 Vertreter Streudiagramme aus der Durchflußzytometrie der CD4 + Zellen und CD8 + Zellen sind in den Abbildungen 4 und 5, bzw. präsentiert. T-Lymphozyten sind weitere identifiziert und phänotypisiert als einer Zellpopulation von dieser Technik. Abbildung 1. Flussdiagramm der kardialen Immunzellen isoliert Verfahren. <img alt="Abbildung 2" src="/files/ftp_upload/3020/3020fig2.jpg" /> . Abbildung 2 Histologische repräsentative Bilder der kardialen Mastzellen sind wie folgt gefärbt: (A) mit Toluidinblau und (B) sind Safranin O / Alcianblau. <img alt="Abbildung 3" src="/files/ftp_upload/3020/3020fig3.jpg" /> Abbildung 3. Functional Studie der kardialen Mastzellen aus der epikardialen Myokard-Region isoliert und stimuliert mit Verbindung 48/80 (10μg/mL) und Substanz P (10 um). <img alt="Abbildung 4" src="/files/ftp_upload/3020/3020fig4.jpg" /> Abbildung 4. Representative Durchflusszytometrie Streudiagramme von isolierten CD4 +-Zellen mit Substanz P (1:100 dilut beschriftetion). <img alt="Abbildung 5" src="/files/ftp_upload/3020/3020fig5.jpg" /> Abbildung 5. Representative Durchflusszytometrie von isolierten CD8 + Zellen mit Substanz P (1:100 Verdünnung) gekennzeichnet.

<ol>
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D., Holland, M., Hauer-Jensen, M., Janicki, J. S., Brower, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protection+from+adverse+myocardial+remodeling+secondary+to+chronic+volume+overload+in+mast+cell+deficient+rats.">Protection from adverse myocardial remodeling secondary to chronic volume overload in mast cell deficient rats.</a> Journal of Molecular and Cellular Cardiology. 43, 56-61 (2008).</li><li>Levick, S. P., McLarty, J. L., Murray, D. B., Freeman, R. M., Carver, W. E., Brower, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+Mast+Cells+Mediate+Left+Ventricular+Fibrosis+in+the+Hypertensive+Rat.">Cardiac Mast Cells Mediate Left Ventricular Fibrosis in the Hypertensive Rat.</a> Hypertension. 53, 1041-1047 (2009).</li><li>Batista, M. L., Santos, R. V., Cunha, L. M., Mattos, K., Oliveira, E. M., Seelander, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+the+pro-inflammatory+cytokine+production+and+peritoneal+macrophage+function+in+rats+with+chronic+heart+failure.">Changes in the pro-inflammatory cytokine production and peritoneal macrophage function in rats with chronic heart failure.</a> Cytokine. 34, 284-290 (2006).</li><li>Galli, S. J., Tsai, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mast+Cells:+Versatile+regulator+of+inflammation,+tissue+remodeling,+host+defense+and+homeostasis.">Mast Cells: Versatile regulator of inflammation, tissue remodeling, host defense and homeostasis.</a> Journal of Dermatological Science. 49, 7-19 (2008).</li><li>Yndestad, A., Holm, A. M., Muller, F., Simonsen, S., Froland, S. S., Gullested, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+expression+of+inflammatory+cytokines+and+activation+markers+in+T-cells+from+patients+with+chronic+heart+failure.">Enhanced expression of inflammatory cytokines and activation markers in T-cells from patients with chronic heart failure.</a> Cardiovasc. Res. 60, 141-146 (2003).</li><li>Bozkurt, B., Kribbs, S. B., Clubb, F. J., Michael, L. H., Didenko, V. V., Hornsby, P. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resident+Cardiac+Mast+Cells+Degranulate+and+Release+Preformed+TNF-&#x0251;,+Initiating+the+Cytokine+Cascade+in+Experimental+Canine+Myocardial+Ischemia/Reperfusion.">Resident Cardiac Mast Cells Degranulate and Release Preformed TNF-&#x0251;, Initiating the Cytokine Cascade in Experimental Canine Myocardial Ischemia/Reperfusion.</a> Circulation. 98, 699-710 (1998).</li><li>Jobe, L. J., Melendez, G. C., Levick, S. P., Du, Y., Brower, G. L., Janicki, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TNF-&#945;+inhibition+attenuates+adverse+myocardial+remodeling+in+a+rat+model+of+volume+overload.">TNF-&#945; inhibition attenuates adverse myocardial remodeling in a rat model of volume overload.</a> Am. J. Physiol. Heart. Circ. Physiol. 297, H1462-H1468 (2009).</li><li>Forman, M. F., Brower, G. L., Janicki, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+histamine+secretion+during+isolation+of+rat+cardiac+mast+cells.">Spontaneous histamine secretion during isolation of rat cardiac mast cells.</a> Inflammation Research. 53, 453-457 (2004).</li><li>Levick, S. P., Murray, D. B., Janicki, J. S., Brower, G. 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Wir haben nichts zu offenbaren.

Dieses Verfahren zur Isolierung von Herz-Immunzellen ermöglicht die Analyse ihrer Funktion, Phänotyp und Reife, ohne die ungewollte Aktivierung, die typischerweise während einer traditionellen enzymatische Verdauung Isolation Technik. Die durchschnittliche Gesamtzahl der Zellen mit dieser Technik erhält man rund 400.000 pro Ratte Herz und Differential-Färbung und Durchflusszytometrie-Analyse dieser Zellen zeigt gemischte Population von T-Lymphozyten (~ 70%), Makrophagen (~ 12%) und Mastzellen mit die Verwendung dieser Technik. (~ 12%). 13, 14 sind daher Reinigungsverfahren für weitere Trennung von Herz Immunsystem Zellpopulationen benötigt. Isolierte Immunzellen kann für mehrere Anwendungen, darunter funktionelle Studien, Durchflusszytometrie, immunofluroescence und Co-Kultur-Experimente genutzt werden. Mit weiteren Reinigung und Experimentieren bieten diese isolierten Zellen ein nützliches Werkzeug für das Verständnis der Rolle des Immunsystems in den Prozess der kardialen Remodeling. Die Ratten wurden unter Standard-Umgebungsbedingungen untergebracht und gepflegt auf kommerzielle Rattenfutter und Leitungswasser ad libitum. Alle Studien zu den Prinzipien des National Institutes of Health Guide für die Pflege und Verwendung von Labortieren angepasst und wurden von der University of South Carolina Institution Animal Care und Verwenden Committee genehmigt. Anästhesie bei der experimentellen Endpunkt wurde von Ketamin / Xylazin-Cocktail bei einem Verhältnis von 2 zu 1 Ketamin Xylazin betroffen. Dies ist ein nonsurvival Verfahren, das die Verwendung von nicht sterilisierten Instrumente ermöglicht. Nach der Isolierung von Immunzellen und anderen gewünschten Gewebe wurde die Ratte schnell und human nach dem American Veterinary Medical Association Guideline zur Euthanasie für Nager (zB Ausbluten, Genickbruch oder Betäubung Überdosierung) eingeschläfert.

<table border="1"><tbody><tr><td> Name des Reagenz und Ausrüstung </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare </td></tr><tr><td> Hanks &#39;Balanced Salt </td><td> Sigma </td><td> H4891 </td><td> Eine Flasche macht 1 L von Hanks &#39;Balance Salz Solution. Rekonstituieren Hanks &#39;Balance Salz Lösung als Teil des Verfahrens. </td></tr><tr><td> Antibiotika-Antimyotic </td><td> Gibco </td><td> 15240-062 </td><td></td></tr><tr><td> HEPES </td><td> Sigma </td><td> H3784 </td><td></td></tr><tr><td> Bovine Serum Albumin </td><td> Sigma </td><td> A3912 </td><td></td></tr><tr><td> 250 mL Nalogene Filter Bottle </td><td> Thermoscientific </td><td> 157-0020 </td><td></td></tr><tr><td> 12 N HCL </td><td> Fisher Scientific </td><td> CAS 7647-01-0 </td><td> Für 6 N, fügen gleichen Volumina von HCL in deionisiertesWasser. </td></tr><tr><td> 10 N NaOH </td><td> Fisher Scientific </td><td> SC267 </td><td> Für 5 N, fügen gleichen Volumina von NaOH zu deionisiertem Wasser. </td></tr><tr><td> Doppelt destilliertem Wasser </td><td></td><td></td><td></td></tr></tbody></table> Tabelle der Ausstattung: <table border="1"><tbody><tr><td> Name des Reagenz und Ausrüstung </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare </td></tr><tr><td> 3-O Suture Silk Braided </td><td> Ethicon Products </td><td> SA64H </td><td></td></tr><tr><td> Small Animal Ventilator </td><td> CWE Incorporated </td><td> SAR-830 </td><td> Set Atemfrequenz auf 40,5 Atemzüge pro min. </td></tr><tr><td> Bevel 18-Gauge-Nadel </td><td> BD Biosciences </td><td> 305199 </td><td> Trim Nadel durch 1 / 2 bis 3 / 4 seiner ursprünglichen Länge für den Einsatz als Trachealtubus. </td></tr><tr><td> Teflon Tip (mit Nadel entfernt) der 24-Gauge-Baxter quick-cath-Katheter </td><td> Baxter </td><td> 2N111 </td><td> Reduzieren Sie die Teflon Tip Länge von 1 / 2 bis 3 / 4. </td></tr><tr><td> 10 cc Spritze </td><td> Fisher Scientific </td><td> 14 bis 823-16B </td><td></td></tr><tr><td> 15 ml Falcon Tubes </td><td> BD Biosciences </td><td> 352097 </td><td></td></tr><tr><td> Sorvall RT6000B Kühlzentrifuge </td><td> Fisher Scientific </td><td> 75-004-377 </td><td> Denken Sie daran, pre-cool und halten bei 4 ° C. </td></tr><tr><td> Hyclone </td><td> Thermoscientific </td><td> S300030.02 </td><td></td></tr><tr><td> Hämozytometer </td><td> Hausser Scientific </td><td> HS-LB-1483 </td><td></td></tr></tbody></table>

isolation of functional cardiac immune cells

Cardiac Immunzellen gewinnen Interesse für die Rollen, die sie spielen in den pathologischen Umbau in vielen Herzerkrankungen 1-5 Diese Immunzellen, die Mastzellen, T-Zellen und Makrophagen gehören,. Speicherung und Freisetzung einer Vielzahl von biologisch aktiven Mediatoren einschließlich Zytokine und Proteasen wie Tryptase. 6-8 Diese Mediatoren haben gezeigt, dass wichtige Akteure in der extrazellulären Matrix Stoffwechsel werden durch die Aktivierung Matrixmetalloproteinasen oder was Kollagenakkumulation durch Modulation der kardialen Fibroblasten-Funktion. 9-11 Allerdings haben verfügbaren Techniken zur Isolierung von Herz-Immunzellen problematisch, weil sie bakterieller Kollagenase, um das Herzmuskelgewebe verdauen verwenden. Diese Technik bewirkt eine Aktivierung der Immunzellen und somit zu einem Verlust der Funktion. Zum Beispiel, kardiale Mastzellen deutlich weniger auf Verbindungen, die Degranulation führen werden. 12 Darum entwickelten wir eine Technik, die für t erlaubtEr Isolation von funktionellen Herz-Immunzellen, die zu einem besseren Verständnis der Rolle dieser Zellen bei Herzerkrankungen führen würde. 13, 14 Diese Methode erfordert eine gewisse Vertrautheit mit der anatomischen Lage der Ratte Xiphoidbasis, Achselhöhle und Lig. falciforme und Perikard des Herzens. Diese Landmarken sind wichtig für den Erfolg des Verfahrens zu erhöhen und eine höhere Ausbeute an Herz-Immunzellen zu gewährleisten. Diese isolierten kardialen Immunzellen können dann für die Charakterisierung der Funktionalität, Erscheinungsbild, Reife und Co-Kultur-Experimente mit anderen Herzzellen zu einem besseren Verständnis ihrer Wechselwirkungen zu gewinnen verwendet werden.

Watch this Scientific Journal Video about Isolation of Functional Cardiac Immune Cells at JoVE.com

Isolation of Functional Cardiac Immune Cells

Diese Methode zur Isolierung von funktionellen Immunzellen aus dem Herzen stellt eine Alternative zu den herkömmlichen Methoden der Kollagenaseverdau, die unerwünschten Immun-Zell-Aktivierung verursacht, was zu einer verminderten Reaktionsfähigkeit dieser Zellen. Unsere Methode der Isolierung ergibt funktionelle Herz-Immunzellen durch die Vermeidung von Problemen mit enzymatische Verdauung.

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 ...

isolation-of-functional-cardiac-immune-cells

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

14.9K Aufrufe.  University of South Carolina- School of Medicine. Diese Methode zur Isolierung von funktionellen Immunzellen aus dem Herzen stellt eine Alternative zu den herkömmlichen Methoden der Kollagenaseverdau, die unerwünschten Immun-Zell-Aktivierung verursacht, was zu einer verminderten Reaktionsfähigkeit dieser Zellen. Unsere Methode der Isolierung ergibt funktionelle Herz-Immunzellen durch die Vermeidung von Problemen mit enzymatische Verdauung.

Serie: Isolation of Functional Cardiac 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

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 ...

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.

Bone marrow transplantation provides a way to change the genotype of the bone marrow derived cells. If the gene of interest is expressed in both bone marrow derived cells and non-bone marrow derived cells, bone marrow transplantation can change the bone marrow derived cells to a different genotype without changing the non-bone marrow derived cell genotype.

Differenziert Functional Roles of Gene Expression von Immun-und Nicht-Immunzellen in Mouse Colitis von Bone Marrow Transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice ...

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 of Adipose Tissue 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 immune cell contribution to local and systemic insulin resistance. Isolation and quantification of different immune cell populations in lean and obese AT is now a commonly utilized technique in immunometabolism laboratories; yet extreme care must be taken both in stromal vascular cell isolation and in the flow cytometry analysis so that the data obtained is reliable and interpretable. In this video we demonstrate how to mince, digest, and isolate the immune cell-enriched stromal vascular fraction. Subsequently, we show how to antibody label macrophages and T lymphocytes and how to properly gate on them in flow cytometry experiments. Representative flow cytometry plots from low fat-fed lean and high fat-fed obese mice are provided. A critical element of this analysis is the use of antibodies that do not fluoresce in channels where AT macrophages are naturally autofluorescent, as well as the use of proper compensation controls.

Adipose tissue (AT) is a site of intense immune cell activation and interaction. Almost all cells of the immune system are present in AT and their ratios are altered by obesity. Proper isolation, quantification, and characterization of AT immune cell populations are critical for understanding their role in immunometabolic disease.

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 ...

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.

Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

Erkennen von Glykogen in peripheren mononukleären Blutzellen mit Perjodsäure-Schiff-Färbung

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

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 ...

Isolation and Characterization of Neutrophil-derived Microparticles for Functional Studies

Polymorphonuclear neutrophil-derived microparticles (PMN)-MPs) are lipid bilayer, spherical microvesicles with sizes ranging from 50&#8211;1,000 nm in diameter. MPs are a newly evolving, important part of cell-to-cell communication and signaling machinery. Because of their size and the nature of their release, until recently MP existence was overlooked. However, with improved technology and analytical methods their function in health and disease is now emerging. The protocols presented here are aimed at isolating and characterizing PMN-MPs by flow cytometry and immunoblotting. Moreover, several implementation examples are given. These protocols for MP isolation are fast, low-cost, and do not require the use of expensive kits. Furthermore, they allow for the labeling of MPs following isolation, as well as pre-labeling of source cells prior to MP release, using a membrane-specific fluorescent dye for visualization and analysis by flow cytometry. These methods, however, have several limitations including purity of PMNs and MPs and the need for sophisticated analytical instrumentation. A high-end flow cytometer is needed to reliably analyze MPs and minimize false positive reads due to noise or auto-fluorescence. The described protocols can be used to isolate and define MP biogenesis, and characterize their markers and variation in composition under different stimulating conditions. Size heterogeneity can be exploited to investigate whether the content of membrane particles versus exosomes is different, and whether they fulfill different roles in tissue homeostasis. Finally, following isolation and characterization of MPs, their function in cellular responses and various disease models (including, PMN-associated inflammatory disorders, such as Inflammatory Bowel Diseases or Acute Lung Injury) can be explored.

New protocols are described here to isolate and characterize microparticles derived from human and mouse neutrophils. These protocols utilize ultracentrifugation, flow cytometry, and immunoblotting techniques to analyze microparticle content, and they can be used to study the role of microparticles derived from various cell types in cellular function.

Isolierung und Charakterisierung von Neutrophilen abgeleitet Mikropartikel für funktionelle Studien

Polymorphonuclear neutrophil-derived microparticles (PMN)-MPs) are lipid bilayer, spherical microvesicles with sizes ranging from 50&#8211;1,000 nm in ...

Isolation of Dendritic Cells from the Human Female Reproductive Tract for Phenotypical and Functional Studies

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low numbers of DCs present per tissue. Yet, as the phenotype and function of DCs is modified by the tissue environment, it is necessary to analyze tissue resident DC populations, since blood derived DCs incompletely reflect the complexities of DCs in tissues. Here we present a protocol to isolate DCs from the human female reproductive tract (FRT) using hysterectomy specimens that allows both phenotypical and functional analyses. The protocol consists of tissue digestion to generate a single cell mixed cell suspension, followed by positive magnetic bead selection. Our tissue digestion protocol does not cleave surface markers, which allows phenotypical and functional analysis of DCs in the steady state, without overnight incubation or cell activation. This protocol can be adapted for the isolation of other immune cell types or isolation of DCs from other tissues.

We describe here a method to isolate and purify dendritic cells from different anatomical compartments in the human female reproductive tract for the evaluation of their phenotypical and functional characteristics. This method can be adapted to isolate other immune cells or dendritic cells from other mucosal tissues.

Isolation von dendritischen Zellen aus menschlichen weiblichen Fortpflanzungsorgane für phänotypische und funktionelle Studien

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low ...