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.

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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 die...

<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

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

15.0K 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

Isolation of Perivascular Multipotent Precursor Cell Populations from Human Cardiac Tissue

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their anatomical tissue location remained unclear. The recent discovery of defined perivascular and MSC cell marker expression by perivascular cells in multiple tissues by our group and other researchers has provided an opportunity to prospectively isolate and purify specific homogenous subpopulations of multipotent perivascular precursor cells. We have previously demonstrated the use of fluorescent activated cell sorting (FACS) to purify microvascular CD146+CD34- pericytes and vascular CD34+CD146- adventitial cells from human skeletal muscle. Herein we describe a method to simultaneously isolate these two perivascular cell subsets from human myocardium by FACS, based on the expression of a defined set of cell surface markers for positive and negative selections. This method thus makes available two specific subpopulations of multipotent cardiac MSC-like precursor cells for use in basic research and/or therapeutic investigations.

Human cardiac tissue harbours multipotent perivascular precursor cell populations that may be suitable for myocardial regeneration. The technique described here allows for the simultaneous isolation and purification of two multipotent stromal cell populations associated with native blood vessels, i.e. CD146+CD34- pericytes and CD34+CD146- adventitial cells, from the human myocardium.

Isolierung von Perivaskuläre multipotenten Vorläuferzellpopulationen aus menschlichem Herzgewebe

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their ...

Forschung

Entwicklungsbiologie

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and membrane ion currents. Those changes are likely&#160;to be responsible for the increased arrhythmogenic risk and the contractile alterations leading to systolic and diastolic dysfunction in cardiac patients. However, most information on the alterations of myocyte function in cardiac diseases has come from animal models.
Here we describe and validate a protocol to isolate viable myocytes from small surgical samples of ventricular myocardium from patients undergoing cardiac surgery operations. The protocol is described in detail. Electrophysiological and intracellular calcium measurements are reported to demonstrate the feasibility of a number of single cell measurements in human ventricular cardiomyocytes obtained with this method.
The protocol reported here can be useful for future investigations of the cellular and molecular basis of functional alterations of the human heart in the presence of different cardiac diseases. Further, this method can be used to identify novel therapeutic targets at cellular level and to test the effectiveness of new compounds on human cardiomyocytes, with direct translational value.

Current knowledge on the cellular basis of cardiac diseases mostly relies on studies on animal models. Here we describe and validate a novel method to obtain single viable cardiomyocytes from small surgical samples of human ventricular myocardium. Human ventricular myocytes can be used for electrophysiological studies and drug testing.

Isolierung und funktionelle Charakterisierung der Menschen ventrikuläre Kardiomyozyten von Fresh Chirurgische Proben

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and ...

Medizin

Isolation and Identification of Extravascular Immune Cells of the Heart

The immune system is an essential component of a healthy heart. The myocardium is home to a rich population of different immune cell subsets with functional compartmentalization both during steady state and during different forms of inflammation. Until recently, the study of immune cells in the heart required the use of microscopy or poorly developed digestion protocols, which provided enough sensitivity during severe inflammation but were unable to confidently identify small &#8212; but key &#8212; populations of cells during steady state. Here, we discuss a simple method combining enzymatic (collagenase, hyaluronidase and DNAse) and mechanical digestion of murine hearts preceded by intravascular administration of fluorescently-labelled antibodies to differentiate small but unavoidable intravascular cell contaminants. This method generates a suspension of isolated viable cells that can be analyzed by flow cytometry for identification, phenotyping and quantification, or further purified with fluorescence-activated cell sorting or magnetic bead separation for transcriptional analysis or in vitro studies. We include an example of a step-by-step flow cytometric analysis to differentiate the key macrophage and dendritic cell populations of the heart. For a medium sized experiment (10 hearts) the completion of the procedure requires 2&#8211;3 h.

This protocol presents a simple and efficient method to isolate, identify and quantify immune cells residing in the myocardium of mice during steady state or inflammation. The protocol combines enzymatic and mechanical digestion for the generation of a single cell suspension that can be further analyzed by flow cytometry.

Isolierung und Identifizierung von Extravascular Immunzellen des Herzens

The immune system is an essential component of a healthy heart. The myocardium is home to a rich population of different immune cell subsets with ...

Immunologie und Infektion

Isolation of Macrophage Subsets and Stromal Cells from Human and Mouse Myocardial Specimens

Macrophages represent the most heterogeneous and abundant immune cell populations in the heart and are central in driving inflammation and reparative responses after cardiac injury. How various subsets of macrophages orchestrate the immune responses after cardiac injury is an active area of research. Presented here is a simple protocol that our lab performs routinely, for the extraction of macrophages from mouse and human myocardium specimens obtained from healthy and diseased individuals. Briefly, this protocol involves enzymatic digestion of cardiac tissue to generate a single cell suspension, followed by antibody staining, and flow cytometry. This technique is suitable for functional assays performed on sorted cells as well as bulk and single cell RNA sequencing. A major advantage of this protocol is its simplicity, minimal day to day variation and wide applicability allowing investigation of macrophage heterogeneity across various mouse models and human disease entities.

Presented here is a protocol to isolate various subsets of macrophages and other non-immune cells from human and mouse myocardium by preparing a single cell suspension through enzymatic digestion. Gating schemes for flow cytometry based identification and characterization of isolated macrophages are also presented.

Isolierung von Makrophagen-Teilmengen und Stromalzellen von Menschlichen und Maus-Myokardproben

Macrophages represent the most heterogeneous and abundant immune cell populations in the heart and are central in driving inflammation and reparative ...

Isolation and Physiological Analysis of Mouse Cardiomyocytes

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force used to pump blood. Individual cardiomyocytes were first isolated over 40 years ago in order to better study the physiology and structure of heart muscle. Techniques have rapidly improved to include enzymatic digestion via coronary perfusion. More recently, analyzing the contractility and calcium flux of isolated myocytes has provided a vital tool in the cellular and sub-cellular analysis of heart failure. Echocardiography and EKGs provide information about the heart at an organ level only. Cardiomyocyte cell culture systems exist, but cells lack physiologically essential structures such as organized sarcomeres and t-tubules required for myocyte function within the heart. In the protocol presented here, cardiomyocytes are isolated via Langendorff perfusion. The heart is removed from the mouse, mounted via the aorta to a cannula, perfused with digestion enzymes, and cells are introduced to increasing calcium concentrations. Edge and sarcomere detection software is used to analyze contractility, and a calcium binding fluorescent dye is used to visualize calcium transients of electrically paced cardiomyocytes; increasing understanding of the role cellular changes play in heart dysfunction. Traditionally used to test drug effects on cardiomyocytes, we employ this system to compare myocytes from WT mice and mice with a mutation that causes dilated cardiomyopathy. This protocol is unique in its comparison of live cells from mice with known heart function and known genetics. Many experimental conditions are reliably compared, including genetic or environmental manipulation, infection, drug treatment, and more. Beyond physiologic data, isolated cardiomyocytes are easily fixed and stained for cytoskeletal elements. Isolating cardiomyocytes via perfusion is an extremely versatile method, useful in studying cellular changes that accompany or lead to heart failure in a variety of experimental conditions.

Individual cardiomyocytes from wild type and mutant mice can be isolated from the heart in order to study their contractility and calcium transients. This allows characterization of the contribution of cellular dysfunction to heart dysfunction from any cause.

Isolation und physiologische Analyse von Maus Kardiomyozyten

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force ...

Biologie

Isolation and Purification of Murine Cardiac Pericytes

Pericytes, perivascular cells of microvessels and capillaries, are known to play a part in angiogenesis, vessel stabilization, and endothelial barrier integrity. However, their tissue-specific functions in the heart are not well understood. Moreover, there is currently no protocol utilizing readily accessible materials to isolate and purify pericytes of cardiac origin. Our protocol focuses on using the widely used mammalian model, the mouse, as our source of cells. Using the enzymatic digestion and mechanical dissociation of heart tissue, we obtained a crude cell mixture that was further purified by fluorescence activating cell sorting (FACS) by a plethora of markers. Because there is no single unequivocal marker for pericytes, we gated for cells that were CD31-CD34-CD45-CD140b+NG2+CD146+. Following purification, these primary cells were cultured and passaged multiple times without any changes in morphology and marker expression. With the ability to regularly obtain primary murine cardiac pericytes using our protocol, we hope to further understand the role of pericytes in cardiovascular physiology and their therapeutic potential.

We have optimized a protocol to isolate and purify murine cardiac pericytes for basic research and investigation of their biology and therapeutic potential.

Isolierung und Reinigung von murinen Herzperizyten

Pericytes, perivascular cells of microvessels and capillaries, are known to play a part in angiogenesis, vessel stabilization, and endothelial barrier ...

Flow Cytometry-Based Quantification and Analysis of Myocardial B-Cells

A growing body of evidence shows that B-lymphocytes play an important role in the context of myocardial physiology and myocardial adaptation to injury. However, the literature reports contrasting data on the prevalence of myocardial B-cells. B-cells have been reported to be both among the most prevalent immune cells in the rodent heart or to be present, but at a markedly lower prevalence than myeloid cells, or to be quite rare. Similarly, several groups have described that the number of myocardial B-cells increases after acute ischemic myocardial injury, but one group reported no changes in the number of B-cells of the injured myocardium. Implementation of a shared, reproducible method to assess the prevalence of myocardial B-cells is critical to harmonize observations from different research groups and thus promote the advancement of the study of B-cell myocardial interactions. Based on our experience, the seemingly contrasting observations reported in the literature likely stem from the fact that murine myocardial B-cells are mostly intravascular and connected to the microvascular endothelium. Therefore, the number of B-cells recovered from a murine heart is exquisitely sensitive to the perfusion conditions used to clean the organ and to the method of digestion used. Here we report an optimized protocol that accounts for these two critical variables in a specific way. This protocol empowers reproducible, flow cytometry-based analysis of the number of murine myocardial B-cells and allows researchers to distinguish extravascular vs. intravascular myocardial B-cells.

Here we report a protocol for the quantification and differentiation of myocardial B-lymphocytes based on their location in the intravascular or endothelial space using flow cytometry.

Durchflusszytometrie-basierte Quantifizierung und Analyse von Myokard-B-Zellen

A growing body of evidence shows that B-lymphocytes play an important role in the context of myocardial physiology and myocardial adaptation to injury. ...

Isolation and Culture of Resident Cardiac Macrophages from the Murine Sinoatrial and Atrioventricular Node

Resident cardiac macrophages have been demonstrated to facilitate the electrical conduction in the heart. The physiologic heart rhythm is initiated by electrical impulses generated in sinoatrial node (SAN) and then conducted to ventricles via atrioventricular node (AVN). To further study the role of resident macrophages in cardiac conduction system, a proper isolation of resident macrophages from SAN and AVN is necessary, but it remains challenging. Here, we provide a protocol for the reliable microdissection of the SAN and AVN in murine hearts followed by the isolation and culture of resident macrophages.
Both, SAN which is located at the junction of the crista terminalis with the superior vena cava, and AVN which is located at the apex of the triangle of Koch, are identified and microdissected. Correct location is confirmed by histologic analysis of the tissue performed with Masson's trichrome stain and by anti-HCN4.
Microdissected tissues are then enzymatically digested to obtain single cell suspensions followed by the incubation with a specific panel of antibodies directed against cell-type specific surface markers. This allows to identify, count, or isolate different cell populations by fluorescent activated cell sorting. To differentiate cardiac resident macrophages from other immune cells in the myocardium, especially recruited monocyte-derived macrophages, a delicate devised gating strategy is needed. First, lymphoid lineage cells are detected and excluded from further analysis. Then, myeloid cells are identified with resident macrophages being determined by high expression of both CD45 and CD11b, and low expression of Ly6C. With cell sorting, isolated cardiac macrophages can then be cultivated in vitro over several days for further investigation. We, therefore, describe a protocol to isolate cardiac resident macrophages located within the cardiac conduction system. We discuss pitfalls in microdissecting and digesting SAN and AVN, and provide a gating strategy to reliably identify, count and sort cardiac macrophages by fluorescence-activated cell sorting.

The protocol presented here provides a step-by-step approach for the isolation of cardiac resident macrophages from the sinoatrial node (SAN) and atrioventricular node (AVN) region of mouse hearts.

Isolierung und Kultur residenter kardialer Makrophagen aus dem murinen sinoatrialen und atrioventrikulären Knoten

Resident cardiac macrophages have been demonstrated to facilitate the electrical conduction in the heart. The physiologic heart rhythm is initiated by ...

Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead myocardium after MI. Although several strategies have been used to regenerate myocardium, stem cell therapy remains a major approach to replenish the dead myocardium of an MI heart. Accumulating evidence suggests the presence of resident cardiac stem cells (CSCs) in the adult heart and their endocrine and/or paracrine effects on cardiac regeneration. However, CSC isolation and their characterization and differentiation toward myocardial cells, especially cardiomyocytes, remains a technical challenge. In the present study, we provided a simple method for the isolation, characterization, and differentiation of CSCs from the adult mouse heart. Here, we describe a density gradient method for the isolation of CSCs, where the heart is digested by a 0.2% collagenase II solution. To characterize the isolated CSCs, we evaluated the expression of CSCs/cardiac markers Sca-1, NKX2-5, and GATA4, and pluripotency/stemness markers OCT4, SOX2, and Nanog. We also determined the proliferation potential of isolated CSCs by culturing them in a Petri dish and assessing the expression of the proliferation marker Ki-67. For evaluating the differentiation potential of CSCs, we selected seven- to ten-days cultured CSCs. We transferred them to a new plate with a cardiomyocyte differentiation medium. They are incubated in a cell culture incubator for 12 days, while the differentiation medium is changed every three days. The differentiated CSCs express cardiomyocyte-specific markers: actinin and troponin I. Thus, CSCs isolated with this protocol have stemness and cardiac markers, and they have a potential for proliferation and differentiation toward cardiomyocyte lineage.

The overall goal of this article is to standardize the protocol for the isolation, characterization, and differentiation of cardiac stem cells (CSCs) from the adult mouse heart. Here, we describe a density gradient centrifugation method to isolate murine CSCs and elaborated methods for CSC culture, proliferation, and differentiation into cardiomyocytes.

Isolierung, Charakterisierung und Differenzierung von kardialen Stammzellen von Erwachsenen Maus Herzen

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead ...

Isolation of Cardiomyocytes from Fixed Hearts for Immunocytochemistry and Ploidy Analysis

The adult mammalian heart is composed of various cell types including cardiomyocytes, endothelial cells and fibroblasts. Since it is difficult to reliably identify nuclei of cardiomyocytes on histological sections, many groups rely on isolating viable cardiomyocytes prior to fixation to perform immunostaining. However, these live cardiomyocyte isolation techniques require optimization to maximize the yield, viability and quality of the samples, with inherent fluctuations from sample to sample despite maximum optimization. Here, we report a reproducible protocol, involving fixation prior to enzymatic digestion of the heart, which leads to maximum yield while preserving the in vivo morphology of individual cardiomyocytes. We further developed an automated analysis platform to determine the number of nuclei and DNA content per nucleus for individual cardiomyocytes. After exposing the chest cavity, the heart was arrested in diastole by perfusion with 60 mM KCl in PBS. Next, the heart was fixed in 4% paraformaldehyde (PFA) solution, and then digested with 60 mg/mL collagenase solution. After digestions, cells were singularized by trituration, and the cardiomyocyte fraction was enriched via differential centrifugation. Isolated cardiomyocytes were stained for Troponin T and &#945;-actinin to assess purity of the obtained population. Furthermore, we developed an image analysis platform to determine cardiomyocyte nucleation and ploidy status following DAPI staining. Image based ploidy assessments led to consistent and reproducible results. Thus, with this protocol, it is possible to preserve native morphology of individual cardiomyocytes to allow immunocytochemistry and DNA content analysis while achieving maximum yield.

The goal of this work is to develop a method to reproducibly isolate cardiomyocytes from the adult heart and measure DNA content and nucleation.

Isolierung von Kardiomyozyten aus festen Herzen für die Immunzytochemie und Ploidie-Analyse

The adult mammalian heart is composed of various cell types including cardiomyocytes, endothelial cells and fibroblasts. Since it is difficult to reliably ...