Diese Arbeit wurde vom National Institutes of Health Award R01 HL105734 zu VVPMDL gelagert ist, zum Teil, von der National Institutes of Health Research Career Award K08 HL094748. VVP ist eine innovative Forscher von der American Heart Association von Grant 11IRG900384 unterstützt.

1. Vorbereitung die notwendigen Lösungen und Anlagen <ol><li> Erstens, die chirurgischen Instrumente zu sterilisieren (gerade Form einer Pinzette, gebogene Form einer Pinzette, Schere gerade Form und gekrümmter Form einer Schere) durch Autoklavieren sie für 15 min. bei 121 ° C aufweist. Bereiten Sie 2 Sets von Instrumenten, wo ein Satz zum Präparieren Sie sich die Herzen und den anderen Satz verwendet wird, zum Zerkleinern das Gewebe verwendet werden. </li><li> Lösen Sie 1 Päckchen MCDB153 Kulturmedien in 900 ml sterilisiertem, gefiltertem Wasser und langsam rühren, bis das Pulver aufgelöst hat. Fügen Sie 1,2 g Natriumbicarbonat, oder 15,7 ml Natriumhydrogencarbonat-Lösung [7,5% w / v], für jeden Liter Gesamtvolumen von Medium vorbereitet und rühren bis gelöst. Den pH-Wert auf 7,2 durch Zugabe von entweder 1 N HCl oder 1 N NaOH, dann gießen Sie das Medium durch einen 0,2 um Flasche Top-Filter unter sterilen Bedingungen in einer Gewebekultur Haube (Biosicherheitswerkbank). Dann sammeln die Medien und speichern sie bei 4 ° C. </li><li> Bereiten Sie die Ergänzungenin den Kulturmedien (Materialliste). Die Zuschläge sollen die Medien unmittelbar vor der Datenträger für die Plattierung der Zellen benötigt hinzugefügt werden. </li><li> Vorbereitung DPBS mit 10% FBS und Antibiotika (Penicillin und Streptomycin). </li><li> Als nächstes bereiten 0,25% Trypsin-Lösung durch Zugabe von 0,25 g Trypsin (Rinderpankreas, Aktivität&gt; 2500 USP Einheiten / mg, hochreines, USB) und 100 ml DPBS und Antibiotika (Penicillin und Streptomycin). </li><li> Weitere benötigte Ausrüstung benötigt, um dieses Verfahren abschließen gehören ein Stereomikroskop, Petrischalen (10 cm), 50 ml konischen Röhrchen, 40-um-Zelle Siebe, DPBS sowie Antibiotika, 60-mm-Kulturschalen und 35-mm-Kulturschalen. </li></ol> 2. Trenn Herzen von Neugeborenen Maus Pups <ol><li> Erstens erhalten P0 bis P2 neonatalen Maus Jungtieren (n = 6 bis 8). Es wird empfohlen, Welpen durch Züchtung Dct-Cre homozygote Weibchen mit Männchen, die heterozygot für entweder die R26R erzeugt verwenden: EYFP Allel oder der Z / EG-Allel, das EtikettMelanozyten-ähnlichen Zellen. Sowohl R26R: EYFP und Z / EG Mäuse sind von Jackson Laboratories (Lager Zahlen 006.148 und 003.920, beziehungsweise). </li><li> Als nächstes einschläfern der Neugeborenen-Maus Welpen, wischen ihre Brust mit einem Alkoholtupfer und legen Sie dann die Welpen in einer 10 cm Petrischale mit DPBS. </li><li> Schneiden Sie die Haut über der Brust und das Brustbein mit sterilisierten Pinzette und Schere und öffnen Sie dann die Brust sorgfältig unter einem Stereomikroskop. Die Herzen der neonatalen Maus Welpen sind sehr klein, so vorsichtig sein, um sicherzustellen, dass die gesamte Herz mit den Vorhöfen angebracht entfernen. </li><li> In DPBS waschen die Herzen und übertragen Sie sie auf einem neuen 10-cm-Petrischale. </li></ol> 3. enzymatische Verdauung <ol><li> Bringen Sie die Petrischale mit den ausgeschnittenen Herzen, um eine Gewebekultur Kapuze (Biosicherheitswerkbank) und folgen Sie dann die folgenden Schritte unter Verwendung von sterilen Techniken in der Kapuze. </li><li> Zunächst dreimal waschen die Herzen in sterilen DPBS und Antibiotika. </li><li> Legen Sie die Herzen (n = 6-8) in ein60-mm-Kulturschale und fügen 6-8 ml 0,5% Trypsin-Lösung in die Schale. </li><li> Weiter schneiden die Herzen in kleine Stücke (von weniger als 1 mm in der Größe) und inkubieren bei 37 ° C für 30 min in einer 5% CO 2 Atmosphäre. </li><li> Nach der Inkubationszeit beendet ist, gieße die resultierende Lösung von Herzgewebe und Trypsin von der Schale in eine sterile 50-ml konischen Röhrchen. </li><li> Dann, mit einem 10-ml sterile Pipette, schnell, aber sanft, verreiben Sie die Lösung in der 50-ml konischen Röhrchen 30-50 mal. </li><li> Filtern der resultierenden Lösung durch ein 40 um-Sieb Zelle auf einem neuen, sauberen 50 ml konischen Röhrchen, um das Filtrat zu sammeln. </li><li> Weiter Zentrifugation der 50-ml-Röhrchen bei 240 x g in einer Tischzentrifuge 5 min bei Raumtemperatur. Dann vorsichtig den Überstand absaugen und Pellet resuspendieren übrigen in 6-8 ml steriler DPBS. Wiederholen Sie diesen Schritt 2 weitere Male. Dieser Schritt Holzpellets die Vorhofmyozyten und Herz Melanozyten aber nicht bringen den fiDa Fibroblasten Fibroblasten sind kleiner und werden im Überstand verbleiben. </li></ol> 4. Auflage Herz Melanozyten-ähnlichen Zellen <ol><li> Nach der dritten Spülung nach der Zentrifugation vorsichtig ablassen oder absaugen der Waschlösung (DPBS) und das Pellet in 6 ml MCDB Kulturmedien, denen die Ergänzungen wurden hinzugefügt. </li><li> Nun ziehen die resuspendierten Lösung in einem sterilen Transferpipette und legen Sie die Zellen in einer 35-mm-Schale oder in einer Vertiefung einer 6-Well-Platte, und bündeln die isolierten Herzzellen von neugeborenen Mäusen 3 in einem 35-mm-Schale oder auch. </li><li> Geschirr Inkubieren über Nacht bei 37 ° C in einem 5% CO 2 Atmosphäre, absaugen dem Kulturmedium zusammen mit allen anhaftende Zellen und dann die Platten einmal gründlich mit DPBS. Dann fügen Sie frisches Medium zu den Platten und ersetzen die Medien alle 2 Tage. Zellen, die aus embryonalen oder neonatalen Herzen isoliert in Kultur für bis zu 3 Wochen beibehalten werden, während Zellen aus erwachsenen hören isoliertts in Kultur für bis zu 10 Tage gehalten werden. </li></ol> 5. Trennherz Melanozyten-ähnlichen Zellen aus adulten Maus Herz <ol><li> Entfernen Sie zunächst die Herzen von reifen Mäuse (3-4 Wochen alt, n = 3) durch eine Mittellinie Sternotomie nach Verabreichung von Heparin 100 U intraperitoneale den Mäusen und dann euthanizing ihnen. Es wird empfohlen, Pentobarbital verwenden, um Mäuse für dieses Verfahren einschläfern, aber dieses Medikament ist nicht an allen Standorten verfügbar. </li><li> Nächsten, waschen Sie die ausgeschnitten Herzen in sterilen DPBS Antibiotika enthalten in einer Gewebekultur Haube (Biosicherheitswerkbank). Drücken Sie die Herzen leicht mit geschwungenen Dissektionszangen, um alle verbleibenden Blut aus ihnen zu entfernen und dann spülen Sie die Herzen in DPBS noch einmal. </li><li> Entfernen Sie den Vorhöfen und AV-Ringraum von den Herzen (diese Strukturen enthalten die Melanozyten-ähnlichen Zellen) und legen Sie die ausgeschnittenen Gewebeproben in einem 35-mm-Schale. </li><li> Schneiden Sie die Herzen in kleine Stücke mit einer gebogenen Schere und Kraftps und fügen 6-8 ml 0,5% Trypsin-Lösung in die Schale. Dann brüten die Gerichte bei 37 ° C für 45-60 min. </li><li> Nun führen Sie die Schritte aus dem oben beschriebenen Verfahren ab Schritt 3.6 ab. </li></ol>

<ol>
	<li>Hornyak, T. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitf+dosage+as+a+primary+determinant+of+melanocyte+survival+after+ultraviolet+irradiation.">Mitf dosage as a primary determinant of melanocyte survival after ultraviolet irradiation.</a> Pigment Cell Melanoma Res. 22, 307-318 (2009).</li><li>Yonetani, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+expansion+of+immature+melanoblasts+and+their+ability+to+repopulate+melanocyte+stem+cells+in+the+hair+follicle.">In vitro expansion of immature melanoblasts and their ability to repopulate melanocyte stem cells in the hair follicle.</a> J. Invest. Dermatol. 128, 408-420 (2008).</li><li>Levin, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Melanocyte-like+cells+in+the+heart+and+pulmonary+veins+contribute+to+atrial+arrhythmia+triggers.">Melanocyte-like cells in the heart and pulmonary veins contribute to atrial arrhythmia triggers.</a> J. Clin. Invest. 119, 3420-3436 (2009).</li><li>Patel, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+insights+in+the+cellular+basis+of+atrial+fibrillation.">Novel insights in the cellular basis of atrial fibrillation.</a> Expert Rev. Cardiovasc. Ther. 8, 907-915 (2010).</li><li>Sviderskaya, E. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+neurons+and+melanocytes+induced+from+immortal+lines+of+postnatal+neural+crest-like+stem+cells.">Functional neurons and melanocytes induced from immortal lines of postnatal neural crest-like stem cells.</a> FASEB J. 23, 3179-3192 (2009).</li><li>Sviderskaya, E. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=P16Ink4a+in+melanocyte+senescence+and+differentiation.">P16Ink4a in melanocyte senescence and differentiation.</a> J. Natl. Cancer. Inst. 94, 446-454 (2002).</li><li>Cook, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+melanoblasts+in+culture:+Expression+of+BRN2+and+synergistic+regulation+by+fibroblast+growth+factor-2,+stem+cell+factor,+and+endothelin-3.">Human melanoblasts in culture: Expression of BRN2 and synergistic regulation by fibroblast growth factor-2, stem cell factor, and endothelin-3.</a> J. Invest. Dermatol. 121, 1150-1159 (2002).</li><li>Fang, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+the+conditions+for+the+generation+of+melanocytes+from+human+embryonic+stem+cells.">Defining the conditions for the generation of melanocytes from human embryonic stem cells.</a> Stem Cells. 24, 1668-1677 (2006).</li><li>Brito, F. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Timeline+and+distribution+of+melanocyte+precursors+in+the+mouse+heart.">Timeline and distribution of melanocyte precursors in the mouse heart.</a> Pigment Cell Melanoma Res. 21, 464-470 (2008).</li><li>Yajima, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+isolation+of+heart+melanocytes+is+specified+and+the+level+of+pigmentation+in+the+heart+may+correlate+with+coat+color.">The isolation of heart melanocytes is specified and the level of pigmentation in the heart may correlate with coat color.</a> Pigment Cell Melanoma Res. 21, 471-476 (2008).</li></ol>

Wir haben nichts zu offenbaren.

Herz Melanozyten sind in anatomischen Stellen innerhalb des Herzens, die häufig Anlass zu Vorhofarrhythmie Trigger vorhanden. Diese Regionen umfassen die Lungenvenen, den atrioventrikulären Annulus, das hintere linke Atrium und das Foramen ovale (Abbildung 4). Um die Rolle dieser Zellen in Arrhythmogenese spielen verstehen, entwickeln wir Techniken, um sie zu isolieren und studieren ihre Physiologie. Jedoch haben wir erkannt, daß eine Verbesserung dieser Technik ergeben gesünder und größeren Menge...

Wir haben vor kurzem identifiziert eine neuartige Zellpopulation im Herzen von Mäusen und Menschen, die Melanin-Synthese Enzyme zu exprimieren und morphologisch ähneln kutanen Melanozyten. Wir als &quot;Herz Melanozyten&quot; Diese Zellen und fanden sie in anatomischen Stellen, an denen Vorhofarrhythmie löst häufig bei Menschen stammen vorhanden sind. Wir fanden auch, Herz Melanozyten dazu beitragen, Vorhofrhythmusstörungen bei Mäusen mit Keimbahn Löschung von DCT. Herz Melanozyten dünn gesamten Maus Vorhof, wo sie weniger als 0,1% aller atrialen Zellen umfassen verteilt. Um die elektrischen und biologischen Eigenschaften von Herz Melanozyten untersuchen wir ein Verfahren, um sie von der Maus mit Herz Cre-induzierbare Melanozyten spezifische Marker zu isolieren entwickelt. Unsere ersten Verfahren wurde von denen zur neonatalen Kardiomyozyten murinen und ergab sehr kleine Zahl von geeigneten für Patch-Clamp-Aufnahmen oder PCR-Analyse entwickelt, Zellen zu isolieren. Jedoch zur Verbesserung der heilenten und die Lebensfähigkeit von Herz Melanozyten, für tiefer gehende Analyse elektrophysiologische oder biochemische Studien entwickelten wir ein verfeinertes Verfahren entwickelt, um von den Melanozyten der Haut zu isolieren. Die beschriebene und in diesem Beitrag gezeigt, Vorgehensweise hat den Vorteil, gesündere Herz Melanozyten, die als reine Population oder mit Kardiomyozyten beschichtet werden können.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comment</td>
 </tr>
 <tr>
 <td>MCDB 153</td>
 <td>Sigma-Aldrich</td>
 <td>M7403</td>
 <td>Dissolve in: Water</td>
 </tr>
 <tr>
 <td>DPBS, no calcium no magnesium</td>
 <td>Invitrogen</td>
 <td>14190136 </td>
 <td>Stock Conc.: 1X 
 Working Conc.: 1X</td>
 </tr>
 <tr>
 <td>Sodium Bicarbonate, or Sodium Bicarbonate Solution, 7.5% </td>
 <td>Sigma-Aldrich </td>
 <td>S5761 or S8761 </td>
 <td>Water 
 Stock Conc.: 1X 
 Working Conc.: 1X</td>
 </tr>
 <tr>
 <td>Penicillin-streptomycin </td>
 <td>Invitrogen</td>
 <td>15140163 </td>
 <td>Stock Conc.: 100X 
 Working Conc.: 1X</td>
 </tr>
 <tr>
 <td>Fetal Bovine Serum</td>
 <td>Life Technology</td>
 <td>Certified</td>
 <td>Working Conc.: 4%</td>
 </tr>
 <tr>
 <td>TPA</td>
 <td>Sigma-Aldrich</td>
 <td>P1585</td>
 <td>DMSO 
 Stock Conc.: 3.2 &mu;M 
 Working Conc.: 32 nM</td>
 </tr>
 <tr>
 <td>Transferrin</td>
 <td>Sigma-Aldrich</td>
 <td>T8158</td>
 <td>DPBS 
 Stock Conc.: 0.1 mg/ml 
 Working Conc.: 1 &mu;g/ml </td>
 </tr>
 <tr>
 <td>Dibutyryl cyclic AMP</td>
 <td>Sigma-Aldrich</td>
 <td>D0627</td>
 <td>DPBS 
 Stock Conc.: 0.5 mM 
 Working Conc.: 5 &mu;M</td>
 </tr>
 <tr>
 <td>Insulin</td>
 <td>Sigma-Aldrich</td>
 <td>I6634</td>
 <td>10 mM 
 acetic acid 
 Stock Conc.: 1 mg/ml 
 Working Conc.: 5 &mu;g/ml</td>
 </tr>
 <tr>
 <td>Basic FGF</td>
 <td>BD Biosciences</td>
 <td>354060</td>
 <td>Water 
 Stock Conc.: 4 ng/&mu;l 
 Working Conc.: 4 ng/ml</td>
 </tr>
 </tbody>
</table>

isolating primary melanocyte-like cells from the mouse heart

Wir identifizierten eine neue Population von Melanozyten-ähnlichen Zellen (auch als Herz Melanozyten) in den Herzen von Mäusen und Menschen, die an Vorhofrhythmusstörungen Trigger in Mäusen bei. Um die elektrischen und biologischen Eigenschaften von Herz Melanozyten untersuchen wir ein Verfahren, um sie von Mäuseherzen, die wir von denen zur neonatalen Kardiomyozyten Maus zu isolieren abgeleitet isolieren entwickelt. Um gesünder Herz Melanozyten geeignet für umfangreichere Patch-Clamp-oder biochemische Untersuchungen zu erhalten, ein raffiniertes Verfahren entwickelt, die wir für die Isolierung und Beschichtung Herz Melanozyten basierend auf den ursprünglich entwickelt, um Haut-Melanozyten zu isolieren. Die raffinierte Vorgehensweise wird in diesem Beitrag gezeigt und produziert eine größere Anzahl von gesunden Melanozyten-ähnlichen Zellen, die als reine Population oder mit Kardiomyozyten beschichtet werden können.

Die in diesem Artikel beschriebene Protokoll ist eine verbesserte Technik im Vergleich zu der, die wir zuvor für die Isolierung Herz Melanozyten von murinen Herz veröffentlicht. Dieses Verfahren setzt die Verwendung von Fluoreszenzmarkern durch Melaninsynthese enzymspezifische Cre-Rekombinase angetriebenen Herz Melanozyten-ähnlichen Zellen zu markieren. Es ist wichtig, um einen Marker zu verwenden, um Herz Melanozyten Etikett beim Arbeiten mit frisch isolierten embryonalen oder neonataler Zellen, da die morphologisch...

Watch this Scientific Journal Video about Isolating Primary Melanocyte-like Cells from the Mouse Heart at JoVE.com

Isolating Primary Melanocyte-like Cells from the Mouse Heart

In diesem Protokoll w e identifiziert eine neue Population von Melanozyten-ähnlichen Zellen (auch als Herz Melanozyten) in den Herzen von Mäusen und Menschen, die an Vorhofrhythmusstörungen löst bei Mäusen bei.

Isolieren Primäre Melanozyten-ähnlichen Zellen aus der Maus Herz

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial ...

isolating-primary-melanocyte-like-cells-from-the-mouse-heart

Research

JoVE Journal

Biology

10.4K Aufrufe.  University of Pennsylvania.  In diesem Protokoll w e identifiziert eine neue Population von Melanozyten-ähnlichen Zellen (auch als Herz Melanozyten) in den Herzen von Mäusen und Menschen, die an Vorhofrhythmusstörungen löst bei Mäusen bei. 

Serie: Isolating Primary Melanocyte-like Cells from the Mouse Heart

Isolating Stem Cells from Soft Musculoskeletal Tissues

Adult stem cells have long been discussed in regards to their application in regenerative medicine. Adult stem cells have generated a great deal of excitement for treating injured and diseased tissues due to their impressive capabilities to undergo multi-lineage cell differentiation and their self-renewal ability. Most importantly, these qualities have made them advantageous for use in autologous cell transplantation therapies. The current protocol will introduce the readers to the modified preplate technique where soft tissues of the musculoskeletal system, e.g. tendon and muscle, are 1st enzymatically dissociated and then placed in collagen coated flasks with medium. The supernatant, which is composed of medium and the remaining floating cells, is serially transferred daily to new flasks. The stem cells are the slowest to adhere to the flasks which is usually takes 5-7 days (serial transfers or preplates) . By using this technique, adult stem cells present in these tissues can be easily harvested through fairly non-invasive procedures.

Isolating adult stem cells from musculoskeletal soft tissues based on the cell's adherence speed to flask.

Isolating Stammzellen aus Soft-Gewebe des Bewegungsapparates

Adult stem cells have long been discussed in regards to their application in regenerative medicine. Adult stem cells have generated a great deal of ...

Forschung

Biologie

Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in unregulated concentrations are detrimental to cell viability1, 2. While living systems have evolved with endogenous and dietary antioxidant defense mechanisms to regulate ROS generation, ROS are produced continuously as natural by-products of normal metabolism of oxygen and can cause oxidative damage to biomolecules resulting in loss of protein function, DNA cleavage, or lipid peroxidation3, and ultimately to oxidative stress leading to cell injury or death4. 
Superoxide radical anion (O2&bull;-) is the major precursor of some of the most highly oxidizing species known to exist in biological systems such as peroxynitrite and hydroxyl radical. The generation of O2&bull;- signals the first sign of oxidative burst, and therefore, its detection and/or sequestration in biological systems is important. In this demonstration, O2&bull;- was generated from polymorphonuclear neutrophils (PMNs). Through chemotactic stimulation with phorbol-12-myristate-13-acetate (PMA), PMN generates O2&bull;- via activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase5. 
Nitric oxide (NO) synthase which comes in three isoforms, as inducible-, neuronal- and endothelial-NOS, or iNOS, nNOS or eNOS, respectively, catalyzes the conversion of L- arginine to L-citrulline, using NADPH to produce NO6. Here, we generated NO from endothelial cells. Under oxidative stress conditions, eNOS for example can switch from producing NO to O2&bull;- in a process called uncoupling, which is believed to be caused by oxidation of heme7 or the co-factor, tetrahydrobiopterin (BH4)8.
There are only few reliable methods for the detection of free radicals in biological systems but are limited by specificity and sensitivity. Spin trapping is commonly used for the identification of free radicals and involves the addition reaction of a radical to a spin trap forming a persistent spin adduct which can be detected by electron paramagnetic resonance (EPR) spectroscopy. The various radical adducts exhibit distinctive spectrum which can be used to identify the radicals being generated and can provide a wealth of information about the nature and kinetics of radical production9.
The cyclic nitrones, 5,5-dimethyl-pyrroline-N-oxide, DMPO10, the phosphoryl-substituted DEPMPO11, and the ester-substituted, EMPO12 and BMPO13, have been widely employed as spin traps--the latter spin traps exhibiting longer half-lives for O2&bull;- adduct. Iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 is commonly used to trap NO due to high rate of adduct formation and the high stability of the spin adduct14.

Electron paramagnetic resonance (EPR) spectroscopy was employed to detect nitric oxide from bovine aortic endothelial cells and superoxide radical anion from human neutrophils using iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)2 and 5,5-dimethyl-1-pyroroline-N-oxide, DMPO, respectively.

Nachweis von Stickstoffmonoxid und Superoxid-Radikalanion von paramagnetischer Resonanzspektroskopie von Zellen mit Spinfallen

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in ...

Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) in the presence of sodium butyrate 1-3. We used this method to reprogram late passage (&gt;p10) human adult fibroblasts derived from Friedreich's ataxia patient (GM03665, Coriell Repository). The reprogramming approach includes highly efficient transduction protocol using repetitive centrifugation of fibroblasts in the presence of virus-containing media. The reprogrammed hiPSC colonies were identified using live immunostaining for Tra-1-81, a surface marker of pluripotent cells, separated from non-reprogrammed fibroblasts and manually passaged 4,5. These hiPSC were then transferred to Matrigel plates and grown in feeder-free conditions, directly from the reprogramming plate. Starting from the first passage, hiPSC colonies demonstrate characteristic hES-like morphology. Using this protocol more than 70% of selected colonies can be successfully expanded and established into cell lines. The established hiPSC lines displayed characteristic pluripotency markers including surface markers TRA-1-60 and SSEA-4, as well as nuclear markers Oct3/4, Sox2 and Nanog. 
The protocol presented here has been established and tested using adult fibroblasts obtained from Friedreich's ataxia patients and control individuals 6, human newborn fibroblasts, as well as human keratinocytes.

We present a protocol for efficient reprogramming of human somatic cells into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) and identification of correctly reprogrammed hiPSC by live staining with Tra-1-81 antibody.

Selektieren und Isolieren Colonies of Human induzierten pluripotenten Stammzellen aus adulten Fibroblasten Umprogrammierte

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding ...

Isolation of Immune Cells from Primary Tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.

Isolierung von Immunzellen aus Primärtumoren

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

Generation der Mäuse aus induzierten pluripotenten Stammzellen

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Protein Isolation from the Developing Embryonic Mouse Heart Valve Region

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis method may not be sufficiently sensitive to detect a protein of interest. To overcome these difficulties, we examined protocols for obtaining protein from adult human cardiac valves and modified these protocols for the developing early embryonic mouse counterparts. In brief, the mouse embryonic aortic valve regions, including the aortic valve and surrounding aortic wall, are collected in the minimal possible volume of a Tris-based lysis buffer with protease inhibitors. If required based on the breeding strategy, embryos are genotyped prior to pooling four embryonic aortic valve regions for homogenization. After homogenization, an SDS-based sample buffer is used to denature the sample for running on an SDS-PAGE gel and subsequent western blot analysis. Although the protein concentration remains too low to quantify using spectrophotometric protein quantification assays and have sample remaining for subsequent analyses, this technique can be used to successfully detect and semi-quantify phosphorylated proteins via western blot from pooled samples of four embryonic day 13.5 mouse aortic valve regions, each of which yields approximately 1 &#956;g of protein. This technique will be of benefit for studying cell signaling pathway activation and protein expression levels during early embryonic mouse valve development.

The analysis of protein expression in young embryonic mouse valves has been hampered by the limited tissue available. This manuscript provides a protocol for preparing protein from developing embryonic mouse valve regions for western blot analysis.

Proteinisolierung aus den Entwicklungs embryonalen Mäuseherzklappen Region

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

Isolation und Primärkultur von Maus Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

Trans-innere Zelle Masse Injektion von embryonalen Stammzellen führt zu höheren Chimerism Raten

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

Primary Cell Cultures from the Mouse Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the eye. It is a single sheet of pigmented cells that are hexagonally packed and connected by tight junctions. The main functions of the RPE include absorption of light, phagocytosis of the shed photoreceptor outer segments, spatial buffering of ions, transport of nutrients, ions and water as well as active involvement in the visual cycle. With such important and diverse functions, it is critically important to study the biology of RPE cells. A number of RPE cell lines have been established; however, passaged and immortalized cells are known to quickly lose some of the morphological and physiological characteristics of natural RPE cells. Thus, primary cells are more suitable for studying different aspects of RPE cell biology and function. Mouse primary RPE cell culture is very useful to researchers since mouse models are widely used in biological studies, however collecting RPE cells from mouse is also very challenging due to their small size. Here, we present a protocol for establishing primary mouse RPE cell cultures which includes enucleation and dissection of the eyes and isolation of the RPE sheets to yield the cells for culturing. This method enables efficient cell recovery. The RPE cells obtained from two mice&#160;can reach confluency on one 12 mm polyester membrane insert pre-loaded in culture plate after one week of culture and display some of the original properties of bona fide RPE cells such as hexagonal shape and pigmentation after two weeks of culture.

The retinal pigment epithelium (RPE) is a multi-functional epithelium of the eye. Here we present a protocol to establish primary cell cultures derived from the murine RPE.

Primären Zellkulturen aus das retinale Pigmentepithel Maus

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the ...

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 ...