Wir möchten Marcelo Toro für die Unterstützung bei der Durchflusszytometrie anerkennen. Diese Studie wurde vom schwedischen Herz-und Lungen-Stiftung, EU-Kommission RP7 &quot;CardioCell&quot;, Schwedischer Forschungsrat, AFA Versicherungen und ALF unterstützt. OB wurde von der Deutschen Forschungsgemeinschaft unterstützt.

1. Die Isolierung des Herz-Kerne <ol><li> Mantel Ultrazentrifuge Rohre (Beckman Zentrifugenröhrchen # 363.664) mit 10 ml 1% BSA / PBS Beschichtungslösung. Verschließen Sie die Röhrchen und lassen Sie sie für 30 min in einem Rohr Rotator drehen. Entfernen Sie die Beschichtungslösung und lassen Sie die Zentrifugenröhrchen Luft trocknen lassen (eine Tube pro Maus Herz für die Analyse der einzelnen Mäuseherzen, abwechselnd bis zu 5 Mäuseherzen oder 1 g Herzgewebe von einer anderen Spezies erforderlich ist (z. B. Mensch) können verarbeitet werden In einem Röhrchen). </li><li> Alle folgenden Schritte sollten auf Eis durchgeführt werden. Präparieren Sie die linke Herzkammer aus frischen oder gefrorenen Snap-Maus-Herz mit einem Skalpell. Man beachte, daß dieses Protokoll für Mäuseherz optimiert, sondern auch von Ratten oder menschlichen Herzens angepasst werden. Alternativ können bis zu 1 g des Herzgewebes von einer anderen Spezies. </li><li> Schneiden Sie die Probe in kleinen Kabinen mit einem Skalpell. </li><li> Übertragen der Gewebestücke in einen 50 ml-Falcon-Röhrchen mit 15 ml Lyse-Puffer gefüllt. </li><li> Homogenisieren derHerzgewebe mit einem T-25 Ultra-Turrax-Homogenisator Sonde (IKA) bei 24.000 UpM für 10 sek. </li><li> Verdünnen des Homogenats mit einem gleichen Volumen Lysepuffer bis 30 ml. </li><li> Verwenden Sie ein Glas Douncer (40 ml) zur weiteren Homogenisierung des Gewebes und die Kerne frei. Führen Sie acht Schläge mit einem großen Abstand Pistill. </li><li> Führen Sie das rohe Kerne durch ein 100 um und 70 um Nylon-Mesh Zellsieb (BD Biosciences), fortlaufend zu isolieren. </li><li> Anzentrifugieren das rohe Kerne in einer gekühlten Zentrifuge (4 ° C) bei 700 × g für 10 min isoliert. </li><li> Entfernen Sie den Überstand vorsichtig durch Umdrehen der Rohre und wischen Sie das Innere des Rohres mit einem Papiertuch. Achten Sie darauf, die Kerne Pellet stören. </li><li> Man löst das rohe Kerne in 5 ml Saccharose-Puffer zu isolieren durch Pipettieren der Lösung mehrmals auf und ab. Fügen Sie eine weitere 25 ml Saccharose-Puffer auf die gelöste Pellet. </li><li> 10 ml frisch zubereiteten Saccharose-Puffer auf die beschichtete Ultrazentrifuge Rohr (siehe Schritt 1.1). &lt;/ Li&gt; <li> Sorgfältig überlagern den zusätzlichen 10 ml Saccharose-Puffer mit dem resuspendierten Pellet Kerne in Saccharose-Puffer von 1,9 Schritt aufgelöst. </li><li> Gleichen Sie die Zentrifugenröhrchen, bevor Sie sie in eine JS13.1 frei schwingenden Rotor und platzieren Sie den Rotor in eine High-Speed-Zentrifuge (Beckman Avanti S-25). </li><li> Drehen der Kerne Probe bei 13.000 × g bei 4 ° C für 60 min. </li><li> Wenn die Spin abgeschlossen ist, entfernen die Röhrchen vorsichtig von dem Rotor und der Überstand verworfen durch Invertieren der Rohre und Abwischen der restlichen Ablagerungen aus dem Inneren der Rohre mit einem Papiertuch. </li><li> Lösen Sie die Kerne Pellet in 1 ml Kerne Pufferspeicher (NSB Plus-Puffer). Hinweis: NSB plus enthält 1,5 mM Spermin als DNA-Stabilisator. </li><li> Fahren Sie mit Schritt 2.1, Immunfärbung von Kardiomyozyten Kernen. </li></ol> 2. Immunfärbung für die Durchflusszytometrie <ol><li> Die negativen Kontrollen für Immunfärbung. Man entnimmt einen aliquoten von 20 ul aus den Kernen Probe und eineDD 980 ul von NSB und Puffer. </li><li> In Anti-pericentriolar Material 1-Antikörper (Kaninchen-Anti-PCM-1, Atlas Antikörper) zu den Kernen Probe in einer Verdünnung von 1:500 bis Immunmarkierung Kardiomyozyten Kernen. In den Isotyp-Antikörper in der gleichen Verdünnung des Anti-PCM-1-Antikörper mit der Negativkontrolle, in Schritt 2.1 hergestellt. </li><li> Inkubieren negativen Kontrolle und Probe-Röhrchen bei 4 ° C über Nacht. </li><li> Waschen Sie Negativ-Kontrolle und Probe mindestens einmal mit NSB Plus-Puffer (Spin-Down Rohre in einer gekühlten Zentrifuge (4 ° C) bei 700 xg für 10 min. Überstand verwerfen und lösen sich die Kerne Pellet in 1 ml NSB Plus-Puffer). </li><li> In Anti-Kaninchen-fluoreszierenden sekundären Antikörper (FITC oder APC) zu negativen Kontrolle und Probenröhrchen in einer Verdünnung von 1:1000. </li><li> Inkubieren negativen Kontrolle und Probe-Röhrchen bei 4 ° C für 1 h. </li><li> Waschen Sie Negativ-Kontrolle und Probe mindestens einmal mit NSB Plus-Puffer (Spin-Down Rohre in einer gekühlten Zentrifuge (4 ° C) bei 700 xg für 10 min. Überstand verwerfen und lösen sich die Kerne Pellet in 1 ml NSB Plus-Puffer). </li><li> Fahren Sie mit der durchflusszytometrischen Analyse und Sortierung. </li></ol> 3. Durchflusszytometrie <ol><li> Coat Kerne Sammlung Röhrchen (Falcon 15 ml) mit 1% BSA / PBS-Lösung, bevor Sie die Durchflusszytometrie Sortierung wie in Schritt 1.1 beschrieben starten. </li><li> Filtern Sie die Probe und die negative Kontrolle durch einen 30 um Zellsieb und laden Sie zuerst die negative Kontrolle der Durchflusszytometer (BD Zufluss). Definieren Sie die erste und zweite Tor zu Kernen und Singuletts (Einzel-Kerne) zu definieren, basierend auf Vorwärtsstreuung (FSC), Vorwärtsstreuung Impulsbreite (FS Pulsbreite) und seitliche Streulicht (SSC) (Abb. 2a und b). Hinzufügen eines DNA-Färbung (DRAQ5 (1:500)) zu der Probe kann helfen, die Kerne Bevölkerung zunächst zu identifizieren. </li><li> Laden der Probe und immunmarkierten definieren das dritte Tor zu Kardiomyozyten Kerne (PCM-1-positve) aus nicht-Kardiomyozyten Kerne (PCM-1-negativen) zu isolieren. Starten Sie die Sortierung(Abb. 2c und d). </li></ol> Optional: Um die Kern-DNA-Gehalt (Ploidie) zu analysieren und Zellzyklus-Analyse durchzuführen fügen Sie eine entsprechende DNA-Färbung der Kerne (zB Hoechst 33342 oder DRAQ5) (Abb. 2e). <ol start="4"><li> Nach der Durchflusszytometrie Sortierung, legen Sie die Kerne auf Eis und neu analysieren, um die Sortierung Reinheit (Abb. 3a und b) zu bestimmen. </li><li> Drehen Sie den sortierten Kerne in den Sammelröhrchen bei 1500 xg in einer Kühlzentrifuge für 15 min. </li><li> Man löst die Kerne Pellets in einer Pufferlösung mit dem Downstream-Anwendungen. </li></ol> 4. Repräsentative Ergebnisse Kerne Morphologie und Integrität durch DNA Flecken beurteilt werden und visualisiert durch Mikroskopie (Abb. 1). Erfolgreiche PCM-1 Kennzeichnung kann durch Epifluoreszenzmikroskopie und mittels Durchflusszytometrie (Abb. 1 und Abb. 2c und d) beurteilt werden. PCM-1-Positive und negative Populationen sollten gut voneinander getrennt werden (Abb. 2c und d). In murinen linken Ventrikel ungefähr 30% aller Kerne Kerne sein sollte Kardiomyozyten (2d). Sortieren Reinheit kann durch neue Analyse der sortierten Kerne (Abb. 3a und b) beurteilt werden. Beide Kerne Bevölkerung sollte eine Sortierung Reinheitsgrad von mehr als 95%. <img alt="1" src="/files/ftp_upload/4205/4205fig1.jpg" /> Abbildung 1. PCM-1 identifiziert Kardiomyozyten Kerne. Cardiac Kerne (a) mit Antikörpern gegen PCM-1 (b) und Nkx2.5 (c) in einer erwachsenen Maus Herzen gefärbt werden. (D) PCM-1-markierte Kerne werden von Kardiomyozyten Zytoplasma (Myosin heavy chain (MHC)) umgeben ist und zum Ausdruck des Transkriptionsfaktors Nkx2.5 und dokumentiert die genaue Identifizierung von Kardiomyozyten Kerne von PCM-1-Färbung (Maßstab 20 pm und 10 um (d, Einschub)). (E) Cardiac Kerne Isolate visualisiert mit dem DNA-Farbstoff DRAQ5. (F und g) CardiomyocYTE Kerne sind mit Antikörpern gegen PCM-1 (Maßstab 10 um) beschriftet. Beachte, dass die epinuclear Färbungsmuster von PCM-1 in Myozyten Kerne im Gewebe aufweist und in isolierten Kernen (Pfeile). <img alt="2" src="/files/ftp_upload/4205/4205fig2.jpg" /> Abbildung 2. Durchflusszytometrische Sortierung von Kardiomyozyten Kernen. (A) Cardiac Kerne werden durch Vorwärtsstreuung (FSC) und Seitwärts-Streulicht (SSC) identifiziert. (B) ein zweites Tor identifiziert einzelne Kerne von FSC und FS Impulsbreite 5. (C, d) Fluoreszenz-Gating ermöglicht die Trennung von Kardiomyozyten Kerne (PCM-1-positiv) und nicht-Kardiomyozyten (PCM-1-negativen) Kerne von Herzgewebe. (E) Maus Kardiomyozyten meist (&gt; 80%) diploid (2n), ist nur eine kleine Teilmenge tetraploiden (4n) 6. Hinweis enthalten menschlichen Kardiomyozyten eine höhere Frequenz von Polyploidie Kerne (&gt; 2n) 7,8. <img alt="Abbildung 3" src="/files/ftp_upload/4205/4205fig3.jpg" /> <strong&gt; Abbildung 3. Reinheit Analyse der sortierten Kardiomyozyten und nicht-Kardiomyozyten Kerne. Re-Analyse der sortierten Nicht-Kardiomyozyten (a) und Kardiomyozyten Kerne (b). Beide Populationen zeigen eine Sortierung Reinheitsgrad von mehr als 99%.

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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+content,+ploidy+level+and+number+of+nuclei+in+the+human+heart+after+myocardial+infarction.">DNA content, ploidy level and number of nuclei in the human heart after myocardial infarction.</a> Cardiovascular Research. 36, 45-51 (1997).</li><li>Adler, C. P., Friedburg, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+DNA+content.+ploidy+level+and+cell+number+in+geriatric+hearts:+postmortem+examinations+of+human+myocardium+in+old+age.">Myocardial DNA content. ploidy level and cell number in geriatric hearts: postmortem examinations of human myocardium in old age.</a> Mol. Cell Cardiol. 18, 3953-39 (1986).</li><li>Dubois, N. 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Vis. Exp. (20), e914 (2008).</li></ol>

Keine Interessenskonflikte erklärt.

Genaue Identifizierung der Kardiomyozyten Kerne ist entscheidend für die Analyse der regenerativen Vorgänge im Myokard 2,3. Herkömmliche Techniken zu Kardiomyozyten aus frischem Gewebe zu isolieren sind hauptsächlich auf enzymatischen Abbau von Proteinen der extrazellulären Matrix und die anschließende Reinigung in der interstitiellen Zellen durch Zentrifugation bei niedriger Geschwindigkeit basiert. Die weitere Reinigung des lebenden Kardiomyozyten aus embryonalen Stammzellen (ESC) Immunmarkierung mit ...

<table border="1"><tbody><tr><td> 1. Lysispuffer </td></tr><tr><td> Name des Reagenzes </td></tr><tr><td> 0,32 M Saccharose </td></tr><tr><td> 10 mM Tris-HCl (pH = 8) </td></tr><tr><td> 5 mM CaCl 2 </td></tr><tr><td> 5 mM Magnesiumacetat </td></tr><tr><td> 2,0 mM EDTA </td></tr><tr><td> 0,5 mM EGTA </td></tr><tr><td> 1 mM DTT </td></tr></tbody></table><table border="1"><tbody><tr><td> 2. Saccharose-Puffer </td></tr><tr><td> Name des Reagenzes </td></tr><tr><td> 2,1 M Saccharose </td></tr><tr><td> 10 mM Tris-HCl (pH = 8) </td></tr><tr><td> 5 mM Magnesiumacetat </td></tr><tr><td> 1 mM DTT </td></tr></tbody></table><table border="1"><tbody><tr><td> 3. Kerne Speicherpuffer (NSB plus) </td></tr><tr&gt; <td> Name des Reagenzes </td></tr><tr><td> 0,44 M Saccharose </td></tr><tr><td> 10 mM Tris-HCl (pH = 7,2) </td></tr><tr><td> 70 mM KCl </td></tr><tr><td> 10 mM MgCl 2 </td></tr><tr><td> 1,5 mM Spermin </td></tr></tbody></table><table border="1"><tbody><tr><td> Chemikalien und Geräte </td><td> Firma </td></tr><tr><td> Kaninchen-IgG-Isotyp-ChIP Grade, # ab37415 </td><td> Abcam </td></tr><tr><td> Kaninchen anti-PCM-1-Antikörper, # HPA023374 </td><td> Atlas Antibodies </td></tr><tr><td> Donkey sek. Antikörper, Anti-Kaninchen Alexa 488 Fluor, # A-21.206 oder gleichwertige sek. Fluoreszenz-Antikörper </td><td> Life Technologies </td></tr><tr><td> DRAQ5 </td><td> Biostatus </td></tr><tr><td> Zelle Siebe 30 um, 70 um und 100 um </td><td> BD Biosciences </td></tr><tr><td> Glass Douncer (40 ml) undPistill &quot;L&quot; </td><td> VWR (Wheaton Industries Inc.) </td></tr><tr><td> T-25 Ultra-Turrax-Homogenisator </td><td> IKA Deutschland </td></tr><tr><td> Dispergierwerkzeug S25 N-18 G </td><td> IKA Deutschland </td></tr><tr><td> Beckman Avanti Zentrifuge </td><td> Beckman Coulter </td></tr><tr><td> Falcon-Tubes 15 ml und 50 ml </td><td> VWR </td></tr><tr><td> Beckman Zentrifugenröhrchen # 363664 </td><td> Beckman Coulter </td></tr><tr><td> JS13.1 frei schwingenden Rotor </td><td> Beckman Coulter </td></tr><tr><td> Influx Zytometer </td><td> Beckman Coulter </td></tr><tr><td> Tube Rotator </td><td> VWR </td></tr></tbody></table>

isolation of cardiomyocyte nuclei from post-mortem tissue

Identifizierung von Kardiomyozyten Kerne hat in Gewebeschnitten Herausforderung gewesen, da die meisten Strategien nur auf zytoplasmatische Markerproteine ​​1 verlassen. Seltene Ereignisse in Herzmuskelzellen wie Proliferation und Apoptose erfordern eine genaue Identifizierung von Kardiomyozyten-Kerne auf die Zellerneuerung in der Homöostase und bei pathologischen Zuständen 2 zu analysieren. Hier stellen wir eine Methode, um Kardiomyozyten Kerne aus post mortem Gewebe zu isolieren durch Sedimentation und Dichte Immunomarkierung mit Antikörpern gegen pericentriolar Material 1 (PCM-1) und anschließende Durchflusszytometrie-Sortierung. Diese Strategie ermöglicht einen hohen Durchsatz-Analyse und Isolation mit dem Vorteil bei der Arbeit ebenso gut auf frischen und gefrorenen Gewebe Archivalien. Dies macht es möglich, das Material bereits in Biobanken gesammelten studieren. Diese Technik ist und die Messung in einer Vielzahl von Arten und mehrfach Downstream-Anwendungen, wie Kohlenstoff-14-3, Zell-cycle-Analyse 4, Visualisierung von Thymidin-Analoga (zB BrdU und IdU) 4, Transkriptom-und epigenetische Analyse.

Watch this Scientific Journal Video about Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue at JoVE.com

Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue

Cardiac Kerne über Dichte Sedimentation isoliert und mit Antikörpern gegen immunmarkiert pericentriolar Material 1 (PCM-1) zu identifizieren und zu sortieren Kardiomyozyten Kerne durch Durchflusszytometrie.

Isolierung von Kardiomyozyten Kerne von Post-mortem Gewebe

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events ...

isolation-of-cardiomyocyte-nuclei-from-post-mortem-tissue

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

16.9K Aufrufe.  University of Lund. Cardiac Kerne über Dichte Sedimentation isoliert und mit Antikörpern gegen immunmarkiert pericentriolar Material 1 (PCM-1) zu identifizieren und zu sortieren Kardiomyozyten Kerne durch Durchflusszytometrie.

Serie: Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue

Isolation of Human Islets from Partially Pancreatectomized Patients

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 diabetic islets from organ donors2. Here we share our protocol for isolating islets from human pancreatic tissue obtained from type 2 diabetic and non-diabetic patients who have undergone partial pancreatectomy due to different pancreatic diseases (benign or malignant pancreatic tumors, chronic pancreatitis, and common bile duct or duodenal tumors). All patients involved gave their consent to this study, which had also been approved by the local ethics committee. The surgical specimens were immediately delivered to the pathologist who selected soft and healthy appearing pancreatic tissue for islet isolation, retaining the damaged tissue for diagnostic purposes. We found that to isolate more than 1,000 islets, we had to begin with at least 2 g of pancreatic tissue. Also essential to our protocol was to visibly distend the tissue when injecting the enzyme-containing media and subsequently mince it to aid digestion by increasing the surface area.
To extend the applicability of our protocol to include the occasional case in which a large amount (&gt;15g) of human pancreatic tissue is available , we used a Ricordi chamber (50 ml) to digest the tissue. During digestion, we manually shook the Ricordi chamber3 at an intensity that varied by specimen according to its level of tissue fibrosis. A discontinous Ficoll gradient was then used to separate the islets from acinar tissue. We noted that the tissue pellet should be small enough to be homogenously resuspended in Ficoll medium with a density of 1.125 g/ml. After isolation, we cultured the islets under stress free conditions (no shaking or rotation) with 5% CO2 at 37 &deg;C for at least 48 h in order to facilitate their functional recovery. Widespread application of our protocol and its future improvement could enable the timely harvesting of large quantities of human islets from diabetic and clinically matched non-diabetic subjects, greatly advancing type 2 diabetes research.

The supply of type 2 diabetic islets for research is insufficient. Here we share our protocol for isolating islets from patients undergoing partial pancreatectomy. This approach represents a unique venue for obtaining islets from type 2 diabetic and clinically matched non-diabetic subjects in adequate numbers for basic and clinical studies.

Isolierung humaner Inselzellen aus teilweise Pankreatektomierten

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 ...

Forschung

Medizin

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

Isolierung von Krebs Stammzellen aus menschlichen Prostatakrebs-Proben

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem cells (ISCs) and progeny that progressively migrate and differentiate toward the tip of the villi. These processes, essential for gastrointestinal homeostasis, have been extensively studied using multiple approaches. Ex vivo technologies, especially primary cell cultures have proven to be promising for understanding intestinal epithelial functions. A long-term primary culture system for mouse intestinal crypts has been established to generate 3-dimensional epithelial organoids. These epithelial structures contain crypt- and villus-like domains reminiscent of normal gut epithelium. Commonly, termed &#8220;enteroids&#8221; when derived from small intestine and &#8220;colonoids&#8221; when derived from colon, they are different from organoids that also contain mesenchyme tissue. Additionally, these enteroids/colonoids continuously produce all cell types found normally within the intestinal epithelium. This in vitro organ-like culture system is rapidly becoming the new gold standard for investigation of intestinal stem cell biology and epithelial cell physiology. This technology has been recently transferred to the study of human gut. The establishment of human derived epithelial enteroids and colonoids from small intestine and colon has been possible through the utilization of specific culture media that allow their growth and maintenance over time. Here, we describe a method to establish a small intestinal and colon crypt-derived system from human whole tissue or biopsies. We emphasize the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

We describe a method to establish human enteroids from small intestinal crypts and colonoids from colon crypts collected from both surgical tissue and biopsies. In this methodological article, we present the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

Gründung des Menschen Epithelial Enteroids und Colonoids von Whole Gewebe und Biopsie

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem ...

Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic uses.
Exosomes have recently been shown to contain microRNAs (miRs). MiRs themselves have arisen as valuable biomarkers for diagnostic purposes. As specimen collection in clinics and hospitals is quite variable, miRNA isolation from whole bile varies substantially. To achieve robust, accurate and reproducible miRNA profiles from collected bile samples in a simple manner required the development of a high-quality protocol to isolate and characterize exosomes from bile. The method requires several centrifugations and a filtration step with a final ultracentrifugation step to pellet the isolated exosomes. Electron microscopy, Western blots, flow cytometry and multi-parameter nanoparticle optical analysis, where available, are crucial characterization steps to validate the quality of the exosomes. For the isolation of miRNA from these exosomes, spiking the lysate with a non-specific, synthetic miRNA from a species like Caenorhabditis elegans, i.e., Cel-miR-39, is important for normalization of RNA extraction efficiency. The isolation of exosome from bile fluid following this method allows the successful miRNA profiling from bile samples stored for several years at -80 &#176;C.

Bile fluid is a valuable source of extracellular vesicles/exosomes that contain potentially important biomarkers. This protocol represents a robust method to isolate exosomes from human bile for further analyses including miRNA profiling.

Isolation und Profilierung von microRNA-haltigen Exosomen von Human Bile

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic ...

Sampling Strategies and Processing of Biobank Tissue Samples from Porcine Biomedical Models

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of genetically modified pig models, and the often-limited number of available animals of these models, establishment of (biobank) collections of adequately processed tissue samples suited for a broad spectrum of subsequent analyses methods, including analyses not specified at the time point of sampling, represent meaningful approaches to take full advantage of the translational value of the model. With respect to the peculiarities of porcine anatomy, comprehensive guidelines have recently been established for standardized generation of representative, high-quality samples from different porcine organs and tissues. These guidelines are essential prerequisites for the reproducibility of results and their comparability between different studies and investigators. The recording of basic data, such as organ weights and volumes, the determination of the sampling locations and of the numbers of tissue samples to be generated, as well as their orientation, size, processing and trimming directions, are relevant factors determining the generalizability and usability of the specimen for molecular, qualitative, and quantitative morphological analyses. Here, an illustrative, practical, step-by-step demonstration of the most important techniques for generation of representative, multi-purpose biobank specimen from porcine tissues is presented. The methods described here include determination of organ/tissue volumes and densities, the application of a volume-weighted systematic random sampling procedure for parenchymal organs by point-counting, determination of the extent of tissue shrinkage related to histological embedding of samples, and generation of randomly oriented samples for quantitative stereological analyses, such as isotropic uniform random (IUR) sections generated by the &#34;Orientator&#34; and &#34;Isector&#34; methods, and vertical uniform random (VUR) sections.

The practical application and performance of methods for generation of representative tissue samples of porcine animal models for a broad spectrum of downstream analyses in biobank projects are demonstrated, including volumetry, systematic random sampling, and differential processing of tissue samples for qualitative and quantitative morphologic and molecular analyses types.

Probenahmestrategien und Verarbeitung der Biobank Gewebeproben von porcinen biomedizinische Modelle

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of ...

Isolation of Primary Human Decidual Cells from the Fetal Membranes of Term Placentae

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized stromal cells and immune cells, are responsible for the secretion of hormonal and inflammatory factors which are critical for successful blastocyst implantation, placental development and play a role in the initiation of labor at term and preterm. Many pregnancy complications can arise from perturbations of a fine balance of different cell populations comprising decidua. Alterations in the proportion of specific decidual cell types may disrupt these crucial processes and increase the risk of developing serious complications of pregnancy, such as embryo implantation failure, intrauterine growth restriction, preeclampsia and preterm labor. The protocol outlined here demonstrates a cost and time effective method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae. By combining enzymatic digestion and gentle mechanical disruption of the decidual tissue, a high yield of decidual cells was obtained with virtually no chorion contamination. Importantly, isolated decidual cells were characterized (stromal cells (55-60%), leukocytes (35%), epithelial (1%) or trophoblast (0.01%) cells) and maintained high viability (80%) which was confirmed by multicolor imaging flow cytometry assay. This protocol is specific to the decidua parietalis and can be adapted to first and second trimester placentae. Once isolated, decidual cells can be used for a multitude of experimental applications aiming to understand the role of different decidual cell sub-populations in pregnancy complications.

This protocol demonstrates a method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae which can be used for a variety of applications (i.e. immunocytochemistry, flow cytometry, etc.) aiming to study the role of different cell populations in pregnancy complications.

Isolation von primären menschlichen Deziduale Zellen aus der fetalen Membranen der Begriff Plazentas

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized ...

Isolation of Atrial Myocytes from Adult Mice

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents responsible for the action potential can cause pro-arrhythmic substrates that underlie arrhythmias, such as atrial fibrillation, which are highly prevalent in many conditions and disease states. Isolating adult mouse atrial cardiomyocytes for use in patch-clamp experiments has greatly advanced our knowledge and understanding of the cellular electrophysiology in the healthy atrial myocardium and in the setting of atrial pathophysiology. In addition, studies using genetic mouse models have elucidated the role of a vast array of proteins in regulating atrial electrophysiology. Here we provide a detailed protocol for the isolation of cardiomyocytes from the atrial appendages of adult mice using a combination of enzymatic digestion and mechanical dissociation of these tissues. This approach consistently and reliably yields isolated atrial cardiomyocytes that can then be used to characterize cellular electrophysiology by measuring action potentials and ionic currents in patch-clamp experiments under a number of experimental conditions.

This protocol is used to isolate single atrial cardiomyocytes from the adult mouse heart using a chunk digestion approach. This approach is used to isolate right or left atrial myocytes that can be used to characterize atrial myocyte electrophysiology in patch-clamp studies.

Isolierung von Atrial Myozyten von erwachsenen Mäusen

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents ...

Electromechanical Assessment of Optogenetically Modulated Cardiomyocyte Activity

Over the past two decades, optogenetic tools have been established as potent means to modulate cell-type specific activity in excitable tissues, including the heart. While Channelrhodopsin-2 (ChR2) is a common tool to depolarize the membrane potential in cardiomyocytes (CM), potentially eliciting action potentials (AP), an effective tool for reliable silencing of CM activity has been missing. It has been suggested to use anion channelrhodopsins (ACR) for optogenetic inhibition. Here, we describe a protocol to assess the effects of activating the natural ACR GtACR1 from Guillardia theta in cultured rabbit CM. Primary readouts are electrophysiological patch-clamp recordings and optical tracking of CM contractions, both performed while applying different patterns of light stimulation. The protocol includes CM isolation from rabbit heart, seeding and culturing of the cells for up to 4 days, transduction via adenovirus coding for the light-gated chloride channel, preparation of patch-clamp and carbon fiber setups, data collection and analysis. Using the patch-clamp technique in whole-cell configuration allows one to record light-activated currents (in voltage-clamp mode, V-clamp) and AP (current-clamp mode, I-clamp) in real time. In addition to patch-clamp experiments, we conduct contractility measurements for functional assessment of CM activity without disturbing the intracellular milieu. To do so, cells are mechanically preloaded using carbon fibers and contractions are recorded by tracking changes in sarcomere length and carbon fiber distance. Data analysis includes assessment of AP duration from I-clamp recordings, peak currents from V-clamp recordings and force calculation from carbon fiber measurements. The described protocol can be applied to the testing of biophysical effects of different optogenetic actuators on CM activity, a prerequisite for the development of a mechanistic understanding of optogenetic experiments in cardiac tissue and whole hearts.

We present a protocol for evaluating the electromechanical effects of GtACR1 activation in rabbit cardiomyocytes. We provide detailed information on cell isolation, culturing and adenoviral transduction, and on functional experiments with the patch-clamp and carbon-fiber techniques.

Elektromechanische Bewertung der optogenetisch modulierten Kardiomyozytenaktivität

Over the past two decades, optogenetic tools have been established as potent means to modulate cell-type specific activity in excitable tissues, including ...

Model of Ischemic Heart Disease and Video-Based Comparison of Cardiomyocyte Contraction Using hiPSC-Derived Cardiomyocytes

Ischemic heart disease is a significant cause of death worldwide. It has therefore been the subject of a tremendous amount of research, often with small-animal models such as rodents. However, the physiology of the human heart differs significantly from that of the rodent heart, underscoring the need for clinically relevant models to study heart disease. Here, we present a protocol to model ischemic heart disease using cardiomyocytes differentiated from human induced pluripotent stem cells (hiPS-CMs) and to quantify the damage and functional impairment of the ischemic cardiomyocytes. Exposure to 2% oxygen without glucose and serum increases the percentage of injured cells, which is indicated by staining of the nucleus with propidium iodide, and decreases cellular viability. These conditions also decrease the contractility of hiPS-CMs as confirmed by displacement vector field analysis of microscopic video images. This protocol may furthermore provide a convenient method for personalized drug screening by facilitating the use of hiPS cells from individual patients. Therefore, this model of ischemic heart disease, based on iPS-CMs of human origin, can provide a useful platform for drug screening and further research on ischemic heart disease.

We present a model of ischemic heart disease using cardiomyocytes derived from human induced pluripotent stem cells, together with a method for quantitative evaluation of tissue damage caused by ischemia. This model can provide a useful platform for drug screening and further research on ischemic heart disease.

Modell der ischämischen Herzkrankheit und videobasierter Vergleich der Kardiomyozytenkontraktion mit hiPSC-abgeleiteten Kardiomyozyten

Ischemic heart disease is a significant cause of death worldwide. It has therefore been the subject of a tremendous amount of research, often with ...

Isolation of Viable Adipocytes and Stromal Vascular Fraction from Human Visceral Adipose Tissue Suitable for RNA Analysis and Macrophage Phenotyping

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release different bioactive molecules that control metabolic, hormonal, and immune processes; currently, it is unclear how these processes are regulated within the adipose tissue. Therefore, the development of methods evaluating the contribution of each cell population to the pathophysiology of adipose tissue is crucial. This protocol describes the isolation steps and provides the necessary troubleshooting guidelines for efficient isolation of viable mature adipocytes and SVF from human VAT biopsies in a single process, using a collagenase enzymatic digestion technique. Moreover, the protocol is also optimized to identify macrophage subsets and perform mature adipocyte RNA isolation for gene expression studies, which allows performing studies dissecting the interaction between these cell populations. Briefly, VAT biopsies are washed, minced mechanically, and digested to generate a single-cell suspension. After centrifugation, mature adipocytes are isolated by flotation from the SVF pellet. The RNA extraction protocol ensures a high yield of total RNA (including miRNAs) from adipocytes for downstream expression assays. Simultaneously, SVF cells are used to characterize macrophage subsets (pro- and anti-inflammatory phenotype) through flow cytometry analysis.

This protocol provides an efficient collagenase digestion method for isolation of viable adipocytes and stromal vascular fraction-SVF cells from human visceral fat in a single process, including methodology to obtain high-quality RNA from adipocytes and phenotypification of SVF-macrophages through staining of multiple membrane-bound markers for analysis by flow cytometry.

Isolierung lebensfähiger Adipozyten und stromaler vaskulärer Fraktion aus menschlichem viszeralem Fettgewebe, geeignet für RNA-Analyse und Makrophagen-Phänotypisierung

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release ...