Die Autoren danken Dr. Paul Witting und Frau Genevieve Fong für ihre Unterstützung bei der mRNA-Extraktion und PCR zu bestätigen, und die Garnett Passe und Rodney Williams Memorial Foundation für die Unterstützung.

Ethik Hinweise: Alle nachstehend beschriebenen Verfahren wurden von der University of Sydney Tierethikkommission genehmigt. 1. Tiere HINWEIS: 1 und 8-9 Monate alten Mäusen (C57BL / 6) aus dem Tier Resources Centre (Perth, Australien) erhalten. Die Mäuse wurden in der Bosch-Nager-Einrichtung an der Universität von Sydney untergebracht. <ol><li> Hausmäuse in Standardmäusekäfige auf einer 12/12 Stunden Licht / Dunkel-Zyklus mit Zugang zu Futter und Wasser ad libitum. </li><li> Teilen Sie Mäusen in jeder Altersgruppe in der Nähe von Infrarot (NIR) behandelt oder scheinbehandelten (Kontroll-) Gruppen zum Vergleich. </li></ol> 2. nahes Infrarot (NIR) Bestrahlung und Sham-Behandlung <ol><li> Rasur der Haare am Kopf und Halsbereich der Maus mit einem elektrischen Rasierer so eng wie möglich, um schnelle Nachwachsen der Haare vor der Beendigung der Behandlungsregime zu verhindern. Um die pathogenfreien Zustand der Mäuse zu erhalten, verwenden Sie 70% EthanolInstrumente zwischen Rasieren Tiere und F10 Veterinärdesinfektionsmittel zwischen Tieren aus getrennten Käfigen reinigen. Tun Sie dies 2-3 Tage vor Beginn der Behandlung, damit Tiere nicht über abgewickelt. </li><li> Restrain Maus, indem das proximale Ende des Schwanzes und lassen Sie ihn in der Handfläche einer Hand oder Tisch entspannen, so dass Stress die zu falschen Ergebnissen führen könnte minimieren. </li><li> Halten Sie die NIR (670 nm) LED (Leuchtdiode) Gerät 1-2 cm von der exponierten (rasiert) Bereich und schalten Sie das Gerät für 90 Sekunden (Abbildung 1A). HINWEIS: Die Temperaturänderung als Ergebnis von 90 Sek Belichtung wurde mit &lt;0,2 ° C in 100 ml Wasser gemessen. </li><li> Wiederholen Sie die Schritte 2,2-2,3 für die Scheinbehandlung Gruppe von Tieren, aber lassen Sie das Gerät ausgeschaltet (Abbildung 1B). </li><li> Wiederholen Sie die Schritte 2,2-2,3 für den NIR-blockiert Behandlung Gruppe von Tieren, sondern decken Sie das Gerät mit Aluminiumfolie. </li><li> Wiederholen Sie die Schritte 2,2-2,5 täglich um approximatE 24-Stunden-Intervallen an 5 aufeinander folgenden Tagen. </li><li> Entpacken Sie die vestibulären Sinnesepithel (3 Cristae und 1 utrikulären Makula) aus beiden Ohren am fünften Tag nach der Behandlung (siehe Abschnitt 3 unten). </li></ol> 3. Die Gewebeextraktion 20 <ol><li> Bereiten 300 ml einer Glycerinbasis künstliche cerebrospinale Flüssigkeit (ACSF), bestehend aus (in mM): 26 NaHCO 3, 11 Glucose, 250 Glycerin, 2,5 KCl, 1,2 NaH 2 PO 4, 1,2 MgCl 2, 2,5 CaCl 2. Vor der Zugabe von CaCl 2, Gas der Lösung mit Carbogen (95% O 2 und 5% CO 2), um einen pH von 7,4 herzustellen und vermeiden Calciumpräzipitation (Trübung). Kühlen Sie die Lösung in einen -80 ° C Gefrierschrank für 45 Minuten, so dass ein Eisbrei gebildet. </li><li> Bereiten Sie die RNA-Isolierung Lysepuffer in markierter Schraubverschluss Mikroröhrchen (stoppt das Knallen der Deckel bei Rohrdruckänderungen zwischen Einfrieren und Auftauen in flüssigem Stickstoff) nachden Anweisungen des Herstellers oder üblichen Laborpraxis. Haben flüssigem Stickstoff fertig in einem Aluminium Dewargefäß. HINWEIS: Verwenden Sie Schutzkleidung und eyeware beim Umgang mit flüssigem Stickstoff. Stellen Sie sicher, dass flüssiger Stickstoff befindet sich in einem gut belüfteten Raum verwendet, da das Volumen der Gasform ist etwa 700 mal so seiner flüssigen Form und können Ersticken verursachen. </li><li> Tief anaesthetize Mäuse mit Ketamin (400 mg / kg) über eine intraperitoneale Injektion. Lassen Sie die Hintergliedmaße Reflex vollständig als Hinweis darauf, dass Mäuse vollständig betäubt abklingen. </li><li> Enthaupten Mäusen mit scharfen Edelstahl Schere und einen Einschnitt entlang der sagittalen Haut des Schädels mit einer Rasierklinge (gerundet # 22). An dieser Stelle und in Schritte von 3,5 bis 3,9, halten das Schädeldach, Gehirn, und die zugrunde liegenden Vestibularapparat so kühl wie möglich durch die regelmäßige Anwendung der eiskalten ACSF über das Gewebe. </li><li> Mit dem spitzen Arm Standardmuster Schere einen small Einschnitt in den Schädel zu Lambda und entlang der Pfeilnaht geschnitten. </li><li> Schieben Sie den Arm von einem flachen Rongeure unter dem Scheitelbein hält die Klinge so nah wie möglich an der unteren Oberfläche des Knochens, ohne Ziehen des Gehirns. Sichern Sie und ziehen weg die Scheitelbein seitlich und das Hinterhauptbein hinten, bis das Gehirn ausgesetzt ist. </li><li> Verwenden Sie einen kleinen Spatel aus rostfreiem Stahl, und heben Sie das Gehirn weg von der vorderen und mittleren Schädelgrube, um die Gleichgewichtsnerv (CN VIII) aus. Durchschneiden den Nerv, um unnötige Spannung auf die primären afferenten Axone, die direkt innervieren die vestibulären Haarzellen zu verhindern. </li><li> Entfernen Sie das Gehirn in toto nach Durchtrennung des KN-VIII. </li><li> Beachten Sie die knöchernen Labyrinth mit der Cochlea und der peripheren vestibulären Organe in der mittleren Schädelgrube. Machen Sie zwei kleine Schnitte neben jeder knöchernen Labyrinth und Verbrauchsteuern die gesamte Struktur, indem Sie den vorderen Bogengangs und später ziehenVerbündeter. </li><li> Sofort tauchen exzidierten Labyrinthe in einer Sezieren Schale mit eiskaltem ACSF-Lösung (wie in Schritt 3.1 beschrieben), während kontinuierlich mit Carbogen Perfusion. </li><li> Unter einem Stereomikroskop, halten Sie durch das Labyrinth der Cochlea und befestigen Sie sie am Boden der Schale mit einer Pinzette. <ol><li> Verwenden Sie gerade feinen Pinzette, eine kleine Öffnung in der Knochen über der vorderen Bogengangs (SSC) Ampulle kratzen. </li><li> Vergrößern Sie vorsichtig diese Öffnung durch das Erreichen unmittelbar unter dem Knochen und schnippte nach außen weg von der Ampulle. Seien Sie vorsichtig, hier nicht schieben Pinzette in die Öffnung und Beschädigung des fragilen häutige Labyrinth unten. Fahren Sie auf diese Weise, bis der Utriculus, vorderen und seitlichen Ampullen alle ausgesetzt sind. Wenn möglich, entfernen Sie auch die hinteren Ampulle. </li></ol></li><li> Mit feinen Pinzette vorsichtig heben Sie den Utriculus und Ampullen aus dem knöchernen Labyrinth, bis sie vollständig gelöst sind. Wo es möglich ist, halten Sie siedurch die damit verbundenen Bogengänge um eine Beschädigung der Sinnesepithel. In einigen Fällen muss der proximale Teil des halbkreisförmigen membranöse Kanal mit Irisschere geschnitten, um die Ampullen aus dem Knochen zu lösen. </li><li> Sicher erfassen die Vestibularorgane zwischen der Spitze der Pinzette und Stelle in den Lysepuffer zuvor in Schritt 3.2 vorbereitet. Vorsichtig schwenken die Zange um im Puffer, um sicherzustellen, Vestibularorgane wurden aus der Zange abgelöst. Überprüfen Sie in diesem Fall, indem die Zange unter dem Stereomikroskop ist. <ol><li> Schrauben Sie den Deckel Mikrotubuli und frieren die Probe in flüssigem Stickstoff unmittelbar. </li></ol></li></ol> 4. RNA-Extraktion und RT-PCR <ol><li> Folgen Standardmethoden der Boten-RNA (mRNA) Extraktion gemäß den Anweisungen des Herstellers oder bevorzugte Laborprotokollen. HINWEIS: Ein handelsüblicher Kit, das Träger RNAs verwendet werden, um zu isolieren geringe Ausbeuten an mRNA wurde in diese verwendetProtokoll. Nieder Elutionsvolumina verwendet, um Endkonzentrationen von mRNA zu erhöhen. HINWEIS: Negative &quot;kein Enzym&quot; Kontrollen (NEC) und &quot;keine DNA-Template&quot; Kontrollen (NTC) müssen ebenfalls durchgeführt werden, um die Gültigkeit der beobachteten Effekte zu gewährleisten. </li><li> Anwenden von Standardverfahren der reversen Transkription von mRNA komplementäre DNA (cDNA) und die Amplifikation von Zielgenen 21-23. </li></ol>

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Die Autoren erklären, sie haben keine konkurrierenden finanziellen Interessen.

Die hier beschriebenen repräsentativen Ergebnisse zeigen, dass kurze transkranielle Lieferung von NIR-Licht (90 sec / Tag für 5 Tage) ist ausreichend, um das Niveau der Antioxidantien Ausdruck in älteren Mäusen zu erhöhen, wenn mit scheinbehandelten Mäusen verglichen. Während abgegebene Wärme könnte eine Quelle der mitochondrialen und / oder neuronale Aktivierung darstellen, wie Ratte berichtet vestibulären Afferenzen 24 - unsere Messung von Wärme durch die NIR emittierten LED-Gerät war &lt;0,2 °...

Degressiven Leistung und anschließende Wasserfälle sind häufig, und leider oft kennzeichnenden Merkmale des natürlichen Alterungs 1. Die Auswirkungen dieser Rückgang ist sowohl physisch als auch sozial sein, und für ältere Menschen deutlich reduziert die Lebensqualität. Als Reaktion darauf haben physikalische Therapie und Rehabilitation im Mittelpunkt der Forschung im Bereich Wasserfälle gewesen, aber noch nicht mit der konsequenten Reduzierung der Prävalenz von wiederholten Stürzen in Verbindung gebracht. Zur gleichen Zeit, Arbeit untersucht Veränderungen im peripheren oder zentralen vestibulären Systems (die für das Gleichgewicht zu halten verantwortlich System) ist knapp, und mögliche therapeutische Strategien zugunsten solcher Systeme und die zugrunde liegenden Ursachen des Ungleichgewichts begrenzt. Jüngste Arbeiten auf altersbedingte neurodegenerative Erkrankungen einschließlich altersbedingter Makuladegeneration 2-4, Alzheimer-Krankheitsmodellen 5-8 und Parkinson 9-12 neuroprotektive Wirkungen si gezeigtenmple nicht-invasive Anwendung von Nahinfrarot (NIR). Ferner wird in dem Gleichgewichtsorgan, NIR verwendet wurde, um die Aktivität der vestibulären primären afferenten Neuronen in vitro 13 zu erhöhen. Während der Mechanismus der NIR-Licht ist nicht gut verstanden, haben die meisten Studien mit NIR vorgeschlagen, dass NIR stimuliert Mitochondrien Komplex IV (Cytochrom-c-Oxidase) 14-17 auf den Zellstoffwechsel zu erleichtern. In der vestibulären Sinnesepithel der subkutanen Platte vom Typ I Haarzellen dicht in Mitochondrien 18 und können als solche einen Wirkort für therapeutische NIR-Behandlung darstellen. Hier eine kurze, nicht-invasive Behandlung Regime transkraniell angewendet NIR, die verwendet werden können, um den Zellstoffwechsel zu messen (und implizit auch die Funktion der Mitochondrien) in der Maus vestibulären Sinnesepithel beschrieben. Ebenfalls diskutiert ist eine Vorbereitung des vestibulären Sinnesepithel und es wird gezeigt, dass NIR erhöht die Expression eines ubiquitous Antioxidans (Superoxiddismutase 1) im Riechepithel - früher gezeigt wichtig für Cochlea Haarzellüberleben 19 zu sein.

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			<td height="63" style="height:63px;">Screw top microtubules</td>
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			<td height="42" style="height:42px;width:216px;">Carbon steel Surgical Blades #22</td>
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			<td height="21" style="height:21px;width:216px;">Friedman-Pearson Rongeurs</td>
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			<td height="42" style="height:42px;width:216px;">Stereo microscope</td>
			<td style="width:101px;">Leica Microsystems</td>
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			<td height="21" style="height:21px;width:216px;">Dumont #5 SF Forceps</td>
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			<td height="21" style="height:21px;width:216px;">Isolate II RNA Micro Kit</td>
			<td style="width:101px;">Bioline</td>
			<td style="width:119px;">BIO-52075</td>
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near infrared (nir) light increases expression of a marker of mitochondrial function in the mouse vestibular sensory epithelium

Strategien zur Dämpfung Rückgang der Abgleichfunktion mit zunehmendem Alter sind überwiegend konzentrierte sich auf physikalische Therapien einschließlich Balance Aufgaben und Bewegung. Allerdings sind diese Ansätze sich nicht auf die zugrunde liegenden Ursachen von Balance Rückgang. Verwendung von Mäusen wurde die Auswirkung von Licht im nahen Infrarot (NIR) auf den Stoffwechsel der Zellen in der vestibulären Sinnesepithel beurteilt. Die gesammelten Daten zeigen, dass diese einfache und sichere Intervention dieser gefährdeten Zellen vor den schädlichen Wirkungen der natürlichen Alterung schützen. mRNA wurde aus der isolierten peripheren vestibulären Sinnesepithel (crista ampullaris und utricular Makula) extrahiert und anschließend in einer cDNA-Bibliothek transkribiert. Diese Bibliothek wurde dann für die Expression von ubiquitären Antioxidans (SOD-1) sondiert. Antioxidans-Genexpression wurde dann verwendet, um den Zellstoffwechsel zu quantifizieren. Mit Hilfe der transkraniellen Lieferung von NIR bei jungen (4 Wochen) und älteren (8-9 Monate) Mäuse und eine kurze Behandlungsregime (90 sec / Tag für 5 days), diese Arbeit legt nahe, NIR allein kann ausreichen, um die Funktion der Mitochondrien im vestibulären Sinnesepithel verbessern. Da gibt es gegenwärtig keine verfügbaren, preiswert, nicht-invasive Therapieverfahren zur vestibulären Haarzellfunktion verbessern, die Anwendung von externen NIR-Strahlung stellt eine mögliche Strategie, um die Auswirkungen der Alterung auf den Zellstoffwechsel inder vestibulären Sinnesepithel entgegenzuwirken.

So vergleichen Sie die Auswirkungen der NIR-Behandlung bei jungen (4 Wochen) und älteren (8-9 Monate) Mäusen messen wir die Expression von antioxidativen Superoxid-Dismutase 1 (SOD-1) in junge (n = 16) und ältere (n = 20) Mäuse, die NIR-behandelten scheinbehandelten oder NIR-blockiert waren. Figur 2 zeigt eine signifikante Zunahme der β-Actin-normalisierten SOD-1-Expression von mehr als 2-fach in jungen NIR-behandelten Tieren im Vergleich zu jungen scheinbehandelten Tieren (p &lt;0,01) und junge NI...

Watch this Scientific Journal Video about Near Infrared NIr Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium at JoVE.com

Near Infrared NIr Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium

Mitochondriale Dysfunktion ist ein Markenzeichen von Zellalterung. Dieser Aufsatz benutzt nicht-invasive nahen Infrarot (NIR) Behandlung die Mitochondrienfunktion im alternden Maus vestibulären Sinnesepithel verbessern.

Nahes Infrarot (NIR) Licht Erhöht Ausdruck einer Markierung der Funktion der Mitochondrien im Maus Vestibular Sinnesepithel

Strategies for attenuating decline in balance function with increasing age are predominantly focused on physical therapies including balance tasks and ...

near-infrared-nir-light-increases-expression-marker-mitochondrial

Research

JoVE Journal

Biology

Near Infrared (NIr) Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium

9.8K Aufrufe.  University of Sydney.  Mitochondriale Dysfunktion ist ein Markenzeichen von Zellalterung. Dieser Aufsatz benutzt nicht-invasive nahen Infrarot (NIR) Behandlung die Mitochondrienfunktion im alternden Maus vestibulären Sinnesepithel verbessern. 

Serie: Near Infrared NIr Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium

Exploring Cognitive Functions in Babies, Children &amp; Adults with Near Infrared Spectroscopy

An explosion of functional Near Infrared Spectroscopy (fNIRS) studies investigating cortical activation in relation to higher cognitive processes, such as language1,2,3,4,5,6,7,8,9,10, memory11, and attention12 is underway worldwide involving adults, children and infants 3,4,13,14,15,16,17,18,19 with typical and atypical cognition20,21,22. The contemporary challenge of using fNIRS for cognitive neuroscience is to achieve systematic analyses of data such that they are universally interpretable23,24,25,26, and thus may advance important scientific questions about the functional organization and neural systems underlying human higher cognition.
Existing neuroimaging technologies have either less robust temporal or spatial resolution. Event Related Potentials and Magneto Encephalography (ERP and MEG) have excellent temporal resolution, whereas Positron Emission Tomography and functional Magnetic Resonance Imaging (PET and fMRI) have better spatial resolution. Using non-ionizing wavelengths of light in the near-infrared range (700-1000 nm), where oxy-hemoglobin is preferentially absorbed by 680 nm and deoxy-hemoglobin is preferentially absorbed by 830 nm (e.g., indeed, the very wavelengths hardwired into the fNIRS Hitachi ETG-400 system illustrated here), fNIRS is well suited for studies of higher cognition because it has both good temporal resolution (~5s) without the use of radiation and good spatial resolution (~4 cm depth), and does not require participants to be in an enclosed structure27,28. Participants cortical activity can be assessed while comfortably seated in an ordinary chair (adults, children) or even seated in mom s lap (infants). Notably, NIRS is uniquely portable (the size of a desktop computer), virtually silent, and can tolerate a participants subtle movement. This is particularly outstanding for the neural study of human language, which necessarily has as one of its key components the movement of the mouth in speech production or the hands in sign language. 
The way in which the hemodynamic response is localized is by an array of laser emitters and detectors. Emitters emit a known intensity of non-ionizing light while detectors detect the amount reflected back from the cortical surface. The closer together the optodes, the greater the spatial resolution, whereas the further apart the optodes, the greater depth of penetration. For the fNIRS Hitachi ETG-4000 system optimal penetration / resolution the optode array is set to 2cm. 
Our goal is to demonstrate our method of acquiring and analyzing fNIRS data to help standardize the field and enable different fNIRS labs worldwide to have a common background.

Here we describe a data collection and data analysis method for functional Near Infrared Spectroscopy (fNIRS), a novel non-invasive brain imaging system used in cognitive neuroscience, particularly in studying child brain development. This method provides a universal standard of data acquisition and analysis vital to data interpretation and scientific discovery.

Exploring Cognitive Functions in Babies, Kinder &amp; Erwachsene mit Nahinfrarot-Spektroskopie

An explosion of functional Near Infrared Spectroscopy (fNIRS) studies investigating cortical activation in relation to higher cognitive processes, such as ...

Forschung

Biologie

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

Mit dem Gene Pulser MXcell Elektroporation System zur Primary Cells mit hohem Wirkungsgrad zu transfizieren

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

Hallo-C: eine Methode, um die dreidimensionale Architektur des Genoms Study.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

Verwendung eines automatisierten Cell Counter zu Genexpressionsstudien Simplify: siRNA Knockdown von IL-4 abhängige Genexpression in Namalwalzellen

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

In vitro Rekonstitution der Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Using High Resolution Computed Tomography to Visualize the Three Dimensional Structure and Function of Plant Vasculature

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being used to evaluate the structure and function of plant xylem network in three dimensions (3D) (e.g. Brodersen et al. 2010; 2011; 2012a,b). HRCT imaging is based on the same principles as medical CT systems, but a high intensity synchrotron x-ray source results in higher spatial resolution and decreased image acquisition time. Here, we demonstrate in detail how synchrotron-based HRCT (performed at the Advanced Light Source-LBNL Berkeley, CA, USA) in combination with Avizo software (VSG Inc., Burlington, MA, USA) is being used to explore plant xylem in excised tissue and living plants. This new imaging tool allows users to move beyond traditional static, 2D light or electron micrographs and study samples using virtual serial sections in any plane. An infinite number of slices in any orientation can be made on the same sample, a feature that is physically impossible using traditional microscopy methods.
Results demonstrate that HRCT can be applied to both herbaceous and woody plant species, and a range of plant organs (i.e. leaves, petioles, stems, trunks, roots). Figures presented here help demonstrate both a range of representative plant vascular anatomy and the type of detail extracted from HRCT datasets, including scans for coast redwood (Sequoia sempervirens), walnut (Juglans spp.), oak (Quercus spp.), and maple (Acer spp.) tree saplings to sunflowers (Helianthus annuus), grapevines (Vitis spp.), and ferns (Pteridium aquilinum and Woodwardia fimbriata). Excised and dried samples from woody species are easiest to scan and typically yield the best images. However, recent improvements (i.e. more rapid scans and sample stabilization) have made it possible to use this visualization technique on green tissues (e.g. petioles) and in living plants. On occasion some shrinkage of hydrated green plant tissues will cause images to blur and methods to avoid these issues are described. These recent advances with HRCT provide promising new insights into plant vascular function.

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique that can be used to study the structure and function of plant vasculature in 3D. We demonstrate how HRCT facilitates exploration of xylem networks across a wide range of plant tissues and species.

Mit hochauflösenden Computertomographie, die dreidimensionale Struktur und Funktion pflanzlicher Gefäßsystem Visualisieren

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being ...

MicroRNA Expression Profiles of Human iPS Cells, Retinal Pigment Epithelium Derived From iPS, and Fetal Retinal Pigment Epithelium

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human iPS cells (iPS-RPE), and fetal RPE. The protocols include collection of RNA for analysis by microarray, and the analysis of microarray data to identify miRNAs that are differentially expressed among three cell types. The methods for culture of iPS cells and fetal RPE are explained. The protocol used for differentiation of RPE from human iPS is also described. The RNA extraction technique we describe was selected to allow maximal recovery of very small RNA for use in a miRNA microarray. Finally, cellular pathway and network analysis of microarray data is explained. These techniques will facilitate the comparison of the miRNA profiles of three different cell types.

The microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human induced-pluripotent stem (iPS) cells (iPS-RPE), and fetal RPE, were compared.

MicroRNA Expressionsprofile der Menschen iPS-Zellen, Retinapigmentepithel Abgeleitet aus iPS und Fetal Retinapigmentepithel

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, ...

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of central nervous system injuries in vivo, possibly by reducing oxidative stress. However, effects of R/NIR-LT on oxidative stress have been shown to vary depending on wavelength or intensity of irradiation. Studies comparing treatment parameters are lacking, due to absence of commercially available devices that deliver multiple wavelengths or intensities, suitable for high through-put in vitro optimization studies. This protocol describes a technique for delivery of light at a range of wavelengths and intensities to optimize therapeutic doses required for a given injury model. We hypothesized that a method of delivering light, in which wavelength and intensity parameters could easily be altered, could facilitate determination of an optimal dose of R/NIR-LT for reducing reactive oxygen species (ROS) in vitro.
Non-coherent Xenon light was filtered through narrow-band interference filters to deliver varying wavelengths (center wavelengths of 440, 550, 670 and 810nm) and fluences (8.5 x 10-3 to 3.8 x 10-1 J/cm2) of light to cultured cells. Light output from the apparatus was calibrated to emit therapeutically relevant, equal quantal doses of light at each wavelength. Reactive species were detected in glutamate stressed cells treated with the light, using DCFH-DA and H2O2 sensitive fluorescent dyes.&#160;
We successfully delivered light at a range of physiologically and therapeutically relevant wavelengths and intensities, to cultured cells exposed to glutamate as a model of CNS injury. While the fluences of R/NIR-LT used in the current study did not exert an effect on ROS generated by the cultured cells, the method of light delivery is applicable to other systems including isolated mitochondria or more physiologically relevant organotypic slice culture models, and could be used to assess effects on a range of outcome measures of oxidative metabolism.

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Non-coherent Xenon light was passed through narrow-band interference and neutral density filters to deliver light of varying wavelength and intensity to cultured cells. This protocol was used to assess the effects of red/near-infrared light therapy on production of reactive species in vitro: no effects were observed using the tested parameters.

Verfahren zur Beurteilung der Auswirkungen einer Reihe von Wellenlängen und Intensitäten von Rot / Nah-Infrarot-Licht-Therapie auf oxidativen Stress<em&gt; In-vitro-</em

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of ...

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Fluorescence microscopy is an indispensable tool for the direct assessment of mitochondrial structure and function in live cells and for studying the mitochondrial structure-function relationship, which is primarily modulated by the molecules governing fission and fusion events between mitochondria. This paper describes and demonstrates specific methods for studying mitochondrial structure and function in live as well as in fixed tissue in the model organism Drosophila melanogaster. The tissue of choice here is the Drosophila ovary, which can be isolated and made amenable for ex vivo live confocal microscopy. Furthermore, the paper describes how to genetically manipulate the mitochondrial fission protein, Drp1, in Drosophila ovaries to study the involvement of Drp1-driven mitochondrial fission in modulating the mitochondrial structure-function relationship. The broad use of such methods is demonstrated in already-published as well as in novel data. The described methods can be further extended towards understanding the direct impact of nutrients and/or growth factors on the mitochondrial properties ex vivo. Given that mitochondrial dysregulation underlies the etiology of various diseases, the described innovative methods developed in a genetically tractable model organism, Drosophila, are anticipated to contribute significantly to the understanding of the mechanistic details of the mitochondrial structure-function relationship and to the development of mitochondria-directed therapeutic strategies.

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Specific methods for studying mitochondrial structure and function in live and fixed Drosophila ovaries are described and demonstrated in this paper.

Studieren Mitochondrial Struktur und Funktion in<em&gt; Drosophila</em&gt; Ovarien

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial ...

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 ...