Les auteurs tiennent à remercier le Dr Paul Witting et Mme Geneviève Fong pour leur aide dans l&#39;extraction de l&#39;ARNm et la PCR, et de la Passe Garnett et Rodney Williams Memorial Foundation pour l&#39;appui.

Déclaration éthique: Toutes les procédures décrites ci-dessous ont été approuvés par l&#39;Université du comité d&#39;éthique animale Sydney. 1. Les animaux NOTE: 1 et 8-9 mois, vieux souris (C57 / BL6) ont été obtenues auprès du Centre des ressources animales (Perth, Australie). Les souris ont été logés dans la facilité Bosch rongeurs à l&#39;Université de Sydney. <ol><li> Maison souris dans des cages standard de la souris sur un 12/12 h cycle lumière / obscurité avec accès à la nourriture et de l&#39;eau ad libitum. </li><li> Divisez souris dans chaque groupe d&#39;âge dans le proche infrarouge (NIR) traités, ou fictive des groupes (de contrôle) traités à titre de comparaison. </li></ol> 2. proche infrarouge (NIR) irradiation et le traitement Sham <ol><li> Rasez la fourrure sur la région de la tête et du cou de la souris avec un rasoir électrique aussi étroitement que possible pour éviter la repousse rapide des cheveux avant la fin du régime de traitement. Afin de maintenir le statut exempt d&#39;agents pathogènes des souris, utiliser 70% d&#39;éthanolpour nettoyer les instruments de rasage entre les animaux et du désinfectant F10 vétérinaire entre les animaux des cages séparées. Pour ce faire, deux à trois jours avant le début du traitement pour que les animaux ne sont pas trop manipulé. </li><li> Retenez la souris en maintenant l&#39;extrémité proximale de la queue et lui permettre de se détendre dans la paume d&#39;une main dessus ou un banc de manière à minimiser le stress qui pourrait conduire à des résultats erronés. </li><li> Tenez le NIR (670 nm) LED (diode électroluminescente) périphérique 1-2 cm de la surface exposée (rasé) et allumer l&#39;appareil pendant 90 secondes (figure 1A). REMARQUE: le changement de température à la suite de l&#39;exposition de 90 secondes a été mesurée comme étant &lt;0,2 ° C dans 100 ml d&#39;eau. </li><li> Répétez les étapes 2.2 à 2.3 pour le groupe d&#39;animaux de traitement de l&#39;imposture, mais laisser l&#39;appareil éteint (figure 1B). </li><li> Répétez les étapes 2.2 à 2.3 pour le groupe de traitement Nir bloqué des animaux, mais couvre l&#39;appareil avec du papier d&#39;aluminium. </li><li> Répétez les étapes 2.2 à 2.5 tous les jours à approximate intervalles de 24 heures pendant 5 jours consécutifs. </li><li> Extraire l&#39;épithélium vestibulaire sensoriel (3 crêtes et une macula utriculaire) des deux oreilles sur le post-traitement cinquième jour (voir la section 3 ci-dessous). </li></ol> 3. Extraction de tissus 20 <ol><li> Préparer 300 ml d&#39;une base de glycérol liquide céphalo-rachidien artificiel (ACSF) consistant en (en mM): 26 NaHCO 3, 11 le glucose, le glycerol 250, KCl 2,5, 1,2 NaH 2 PO 4, 1,2 MgCl 2, CaCl 2 et 2,5. Avant l&#39;addition de CaCl 2, le gaz de la solution avec du carbogène (95% O 2 et 5% CO 2) pour établir un pH de 7,4 et éviter la précipitation de calcium (turbidité). Refroidir la solution dans un congélateur à -80 ° C pendant 45 minutes de sorte qu&#39;un coulis de glace est formée. </li><li> Préparer le tampon de lyse d&#39;isolement d&#39;ARN dans des microtubes meilleurs vis marqués (arrête l&#39;éclatement de couvercles lorsque les changements de pression du tube entre gel et dégel dans l&#39;azote liquide) selonaux instructions du fabricant ou les pratiques de laboratoire habituelles. Avoir l&#39;azote liquide prêt dans un flacon en aluminium de Dewar. REMARQUE: Utiliser des vêtements de protection et eyeware lors de la manipulation de l&#39;azote liquide. Assurez-vous que l&#39;azote liquide est utilisé dans une pièce bien aérée que le volume de sa forme de gaz est d&#39;environ 700 fois supérieur à celui de sa forme liquide et peut causer l&#39;asphyxie. </li><li> Profondément anesthésier les souris avec la kétamine (400 mg / kg) par une injection intra-péritonéale. Laisser le réflexe des membres postérieurs se calmer complètement comme une indication que les souris sont pleinement anesthésiés. </li><li> Décapiter souris avec des ciseaux pointus en acier inoxydable et faire une incision le long de la peau sagittale du crâne à l&#39;aide d&#39;une lame de rasoir (arrondi # 22). À ce stade et les étapes tout au long de 3.5 à 3.9, gardez la voûte crânienne, le cerveau et appareil vestibulaire sous-jacente aussi frais que possible par l&#39;application régulière de la glace-froid ACSF sur le tissu. </li><li> En utilisant le bras de ciseaux pointus de modèle standard faire un small incision dans le crâne au Lambda et couper le long de la suture sagittale. </li><li> Glisser doucement un bras de rongeurs peu profondes sous l&#39;os pariétal maintien de la lame le plus près possible de la surface inférieure de l&#39;os sans glisser le cerveau. Fixez et retirez l&#39;os pariétal latéralement et l&#39;os occipital en arrière jusqu&#39;à ce que le cerveau est exposé. </li><li> Utilisez une petite spatule en acier inoxydable et soulevez le cerveau loin de la partie antérieure et fosse cérébrale moyenne pour exposer le nerf vestibulaire (CN VIII). Transect le nerf pour éviter des tensions inutiles sur les axones afférents primaires qui innervent directement les cellules ciliées vestibulaires. </li><li> Retirer le cerveau in toto après transection du CN VIII. </li><li> Observez le labyrinthe osseux contenant la cochlée et les organes vestibulaires périphériques dans la fosse cérébrale moyenne. Faire deux petites incisions à côté de chaque labyrinthe osseux et exciser toute la structure en tenant le canal semi-circulaire antérieure et en tirant plus tardallié. </li><li> Immerger immédiatement labyrinthes excisées dans un plat contenant dissection solution ACSF glacée (comme décrit dans l&#39;étape 3.1) tout en perfusion continue avec carbogène. </li><li> Sous un stéréomicroscope, maintenez le labyrinthe par la cochlée et le fixer au fond du plat avec une pince. <ol><li> Utilisez une pince droite fines rayer une petite ouverture dans l&#39;os au-dessus du canal semi-circulaire (SSC) ampoule antérieure. </li><li> Agrandir doucement cette ouverture en atteignant immédiatement sous l&#39;os et pichenette vers l&#39;extérieur de l&#39;ampoule. Soyez prudent ici pour ne pas pousser pince dans l&#39;ouverture et endommager le labyrinthe membraneux fragile ci-dessous. Continuez de cette façon jusqu&#39;à ce que le utricule, antérieure et latérale ampoules sont tous exposés. Si possible enlever l&#39;ampoule postérieure aussi. </li></ol></li><li> En utilisant des pinces fines, soulevez doucement le utricule et ampoules loin du labyrinthe osseux jusqu&#39;à ce qu&#39;ils soient complètement détachés. Lorsque cela est possible, les tenirpar leurs canaux semi-circulaires associés pour éviter d&#39;endommager l&#39;épithélium sensoriel. Dans certains cas, la partie proximale du canal semi-circulaire membraneuse peut être nécessaire de couper avec des ciseaux iris pour libérer les ampoules de l&#39;os. </li><li> Saisir fermement les organes vestibulaires entre la pointe de pince et le lieu dans le tampon de lyse préparé plus tôt dans l&#39;étape 3.2. Remuer doucement la pince autour dans le tampon pour assurer organes vestibulaires ont détaché de la pince. Voir ce est le cas en amenant les pinces au microscope stéréoscopique. <ol><li> Visser sur le couvercle de microtubules et de geler l&#39;échantillon dans de l&#39;azote liquide immédiatement. </li></ol></li></ol> 4. Extraction de l&#39;ARN et RT-PCR <ol><li> Suivre les méthodes standard de l&#39;ARN messager (ARNm) extraction selon les instructions du fabricant ou les protocoles de laboratoire préférés. REMARQUE: Un kit commercial qui a utilisé ARN porteurs pour aider à isoler de petits rendements de l&#39;ARNm a été utilisé dans ceprotocole. Volumes d&#39;élution plus basses peuvent être utilisées pour augmenter les concentrations finales de l&#39;ARNm. NOTE: Négatif contrôles &quot;pas d&#39;enzymes&quot; (CNE) et des contrôles &quot;pas de modèle d&#39;ADN» (NTC) doivent également être remplies pour assurer la validité des effets observés. </li><li> Appliquer les méthodes standard de la transcription inverse de l&#39;ARNm en ADN complémentaire (ADNc) et l&#39;amplification de gènes cibles de 21 à 23. </li></ol>

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Les auteurs déclarent qu&#39;ils ne ont pas des intérêts financiers concurrents.

Les résultats représentatifs décrits ici montrent que brève prestation transcrânienne de la lumière NIR (90 sec / jour pendant 5 jours) est suffisant pour élever les niveaux d&#39;expression antioxydant chez les souris âgées par rapport aux souris sham. Alors que la chaleur émise pourrait représenter une source de mitochondrial et / ou l&#39;activation neuronale, tels que déclarés pour vestibulaire de rat afférences 24 - notre mesure de la chaleur émise par le NIR LED dispositif était &lt;0,2 ...

Baisse du rendement de l&#39;équilibre et chutes ultérieures sont communs, et les caractéristiques définissant malheureusement souvent de vieillissement naturel 1. L&#39;impact de cette baisse peut être à la fois physique et social, et réduit considérablement la qualité de vie des personnes âgées. En réponse, les thérapies physiques et de réhabilitation ont fait l&#39;objet de la recherche sur les chutes, mais ne ont pas été associés à une réduction constante de la prévalence des chutes répétées. Dans le même temps, le travail d&#39;instruction des changements dans le système vestibulaire périphérique ou central (le système chargé de maintenir l&#39;équilibre) est rare, et les stratégies thérapeutiques potentielles ciblant ces systèmes et les causes sous-jacentes du déséquilibre limitée. Des travaux récents sur les troubles neurodégénératifs liés à l&#39;âge, y compris liée à l&#39;âge, la dégénérescence maculaire 2-4 modèles de la maladie d&#39;Alzheimer 5-8, et la maladie de Parkinson 9-12 ont montré des effets neuroprotecteurs de simple application non invasive du proche infrarouge (NIR) de lumière. En outre, dans le système vestibulaire, NIR a été utilisé pour augmenter l&#39;activité des neurones afférents primaires vestibulaires 13 in vitro. Bien que le mécanisme de la lumière NIR ne est pas bien compris, la plupart des études par NIR ont suggéré que Nir stimule les mitochondries complexe IV (cytochrome c oxydase) 14-17 pour faciliter le métabolisme cellulaire. Dans l&#39;épithélium vestibulaire sensoriel la plaque sous-cutanée de cellules Je cheveux de type est dense dans les mitochondries 18 et en tant que telle peut représenter un site d&#39;action pour le traitement Nir thérapeutique. Ici, un bref régime de traitement non invasif des transcranially appliqué Nir qui peut être utilisé pour mesurer le métabolisme cellulaire (et par voie de conséquence, la fonction mitochondriale) dans l&#39;épithélium vestibulaire sensoriel de la souris est décrit. En outre examiné est une préparation de l&#39;épithélium sensoriel vestibulaire et il est montré que NIR augmente l&#39;expression d&#39;un ubiquitonous antioxydant (superoxyde dismutase 1) dans l&#39;épithélium sensoriel - précédemment révélé important pour les cheveux de la survie cellulaire 19 cochlée.

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			<td height="42" style="height:42px;">Quantum WARP 10</td>
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			<td height="63" style="height:63px;">Screw top microtubules</td>
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			<td height="63" style="height:63px;width:216px;">Ketamine</td>
			<td style="width:101px;">Parnell, Alexandria Australia</td>
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			<td height="21" style="height:21px;">Standard Pattern Scissors</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 style="width:119px;">16221-14</td>
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			<td height="42" style="height:42px;width:216px;">Stereo microscope</td>
			<td style="width:101px;">Leica Microsystems</td>
			<td style="width:119px;">A60S</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dumont #5 SF Forceps</td>
			<td style="width:101px;">FST</td>
			<td style="width:119px;">11252-00</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

Stratégies d&#39;atténuation déclin de la fonction de balance avec l&#39;âge sont principalement ciblée sur les thérapies physiques y compris les tâches de l&#39;équilibre et de l&#39;exercice. Cependant, ces approches ne traitent pas les causes sous-jacentes du déclin de l&#39;équilibre. En utilisant des souris, l&#39;impact de la lumière proche infrarouge (NIR) sur le métabolisme des cellules de l&#39;épithélium vestibulaire sensoriel a été évaluée. Les données recueillies montrent que cette intervention simple et sûr peut protéger ces cellules vulnérables contre les effets délétères du vieillissement naturel. L&#39;ARNm a été extrait à partir de l&#39;épithélium sensoriel vestibulaire périphérique isolé (crête ampullaire et macule utriculaire) et ensuite transcrite en une banque d&#39;ADNc. Cette banque a été ensuite sondée pour l&#39;expression ubiquiste d&#39;antioxydant (SOD-1). L&#39;expression du gène antioxydant a ensuite été utilisé pour quantifier le métabolisme cellulaire. Utilisation de la livraison transcrânienne de NIR chez les jeunes (4 semaines) et plus âgés (8-9 mois) des souris, et un régime de traitement brève (90 secondes / jour pendant 5 joursays), ce travail suggère Nir peut suffire à améliorer la fonction mitochondriale dans l&#39;épithélium vestibulaire sensoriel. Comme il n&#39;y a pas de méthodes disponibles, abordables et non invasives de la thérapie pour améliorer la fonction des cellules ciliées vestibulaires, l&#39;application d&#39;un rayonnement NIR externe fournit une stratégie potentielle pour contrer l&#39;impact du vieillissement sur le métabolisme cellulaire INTHE épithélium vestibulaire sensoriel.

Pour comparer l&#39;impact du traitement NIR chez les jeunes (4 semaines) et plus âgés (8-9 mois) des souris, nous avons mesuré l&#39;expression d&#39;antioxydant superoxyde dismutase 1 (SOD-1) chez les jeunes (n = 16) et plus (n = 20) souris qui étaient Nir-traitée, sham-traitée, ou NIR bloqué. Figure 2 montre une augmentation significative de la β-actine normalisée SOD-1 expression de plus de 2 fois chez les jeunes Nir-animaux traités par rapport aux jeunes animaux sham (p &lt;0,01) et les j...

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

Dysfonction mitochondriale est une caractéristique de la sénescence cellulaire. Ce document utilise le proche infrarouge (NIR) traitement non-invasif pour améliorer la fonction mitochondriale chez la souris épithélium vestibulaire sensoriel vieillissement.

Proche infrarouge (NIR) Lumière augmente l&#39;expression d&#39;un marqueur de mitochondrial Fonction chez la souris vestibulaire sensoriel épithélium

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 vues.  University of Sydney.  Dysfonction mitochondriale est une caractéristique de la sénescence cellulaire. Ce document utilise le proche infrarouge (NIR) traitement non-invasif pour améliorer la fonction mitochondriale chez la souris épithélium vestibulaire sensoriel vieillissement. 

Vidéo: 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.

L&#39;exploration des fonctions cognitives chez les bébés, enfants et adultes avec la spectroscopie proche infrarouge

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

Recherche

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.

Utilisation du système d&#39;électroporation Gene Pulser MXcell à transfecter des cellules primaires à haut rendement

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.

Salut-C: une méthode d&#39;étude de l&#39;architecture tridimensionnelle des génomes.

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.

Aide d&#39;un compteur de cellules automatisé pour simplifier les études d&#39;expression génique: Knockdown siRNA de l&#39;IL-4 dans l&#39;expression des gènes dépendant cellules Namalwa

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.

La reconstitution in vitro de l&#39;actif T. La télomérase castaneum

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.

Utilisation à haute résolution tomodensitométrie pour visualiser la structure tridimensionnelle et la fonction des vaisseaux sanguins des végétaux

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.

Profils MicroRNA d&#39;expression de cellules iPS humaines, l&#39;épithélium pigmentaire rétinien Derived From iPS, et fœtale épithélium pigmentaire rétinien

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.

Méthode d&#39;évaluation des effets d&#39;une gamme de longueurs d&#39;onde et des intensités de Luminothérapie Rouge / proche infrarouge sur le stress oxydatif<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.

Étudier mitochondrial Structure et fonction<em&gt; Drosophila</em&gt; ovaires

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.

Cultures de cellules primaires de l’épithélium pigmentaire rétinien de souris

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