Les auteurs tiennent à remercier John Woolford pour la première base de cette méthode, TGK est soutenu par les NIH et GM057483 AI076245 et PRC est soutenu par GM77073.

1. Préparation des gradients de sucrose 7-47% <ol><li> Préparer des gradients de sucrose un jour avant l&#39;utilisation pour permettre aux gradients de devenir permanente. Faire stériles 7% et 47% de saccharose des solutions contenant 50 mM de NH 4 Cl, 7,0 50mM pH Tris-Acétate et 12mm MgCl 2 et stocker à 4 ° C 1. </li><li> Pour 6% des gradients de mélanger les 7 et 47% de saccharose solutions de stock pour faire 48 ml de chacune des concentrations de saccharose suivantes. Ajouter la TNT (1M stock) à chaque solution de saccharose à une concentration finale de 1 mM. <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> Concentration finale </td><td> 7% de saccharose </td><td> 47% de saccharose </td></tr><tr><td> 7% </td><td> 48 ml </td><td> 0 </td></tr><tr><td> 17% </td><td> 36 ml </td><td> 12 ml </td></tr><tr><td> 27% </td><td> 24 ml </td><td> 24 ml </td></tr><tr><td> 37% </td><td> 12 ml </td><td> 36 ml </td></tr><tr><td> 47% </td><td> 0 </td><td> 48 ml </td></tr></tbody></table></li><li> Afin de verser les gradients, joignez une pipette Pasteur pour une seringue de 20 ml par la sécurisation et d&#39;étanchéité avec Parafilm ou l&#39;utilisation d&#39;une aiguille longue. Ajouter 7 ml de saccharose à 7% au fond d&#39;un 1 x 3.5 &quot;tube d&#39;ultracentrifugation polyallomère. Suivant ajouter 7 ml de solution de saccharose 17% dans la solution de 7% en plaçant la pointe pipette Pasteur près du fond du tube et de pipetage pas plus vite que 0,3 ml / sec. Répéter avec 7 ml chacun de 27%, 37% et des solutions de saccharose de 47%. Vous devez respecter les lignes claires entre les couches, ce qui indique que le mélange minimale est survenue. gradients de magasin à 4 ° C pendant la nuit avec les rotors, les bouteilles et des tubes qui seront utilisés pour récolter des échantillons. </li></ol> 2. Préparation de l&#39;extrait de levure <ol><li> Cultivez 125 ml de culture de levure à une DO 600 de 0,8 à 1,0. Ajouter cycloheximide à la culture à une concentration finale de 100 pg / ml et continuer agitation à 30 ° C pendant 15 min. </li><li> Pendant ce temps, préparer un tampon de lyse contenant 80 pg / ml de cycloheximide, 200 pg / ml d&#39;héparine, DEPC 0,2%, et 10 mM Tris-HCl pH 7,5, 0,1 M de NaCl, 30 mM MgCl 2. Gardez tout sur la glace à partir de ce point. </li><li> Verser la culture dans un flacon de 250 ml à centrifuger et remplir de glace. Centrifuger à 8000 xg (7250 rpm dans un rotor SLA-1500) pendant 5 min à 4 ° C. Laver le culot une fois avec 10 ml de tampon de lyse de la glace froide et le transfert des tubes de 15 ml en polypropylène. Centrifuger à 5900 xg (~ 6000 rpm dans un rotor SLA-600TC) pendant 3 min à 4 ° C. Reprendre le culot dans 0,5 ml de tampon de lyse, le transfert au tube 1.5ml et ajoutez 0,5 ml d&#39;réfrigérés, cuits perles de verre lavée à l&#39;acide. Vortex les cellules pour des intervalles de quatre secondes 30, le tube de refroidissement sur la glace pendant 30 secondes entre chaque intervalle. Ajouter une autre dose de 0,5 mL de tampon de lyse dans le tube de 1,5 ml. Centrifuger à vitesse maximale dans une microcentrifugeuse (14 000 rpm dans un microcentrifugeuse Eppendorf 5417R) pendant 10 min à 4 ° C. Transférer le surnageant dans un nouveau tube de 1,5 ml microtubes. Retirer 10 ul d&#39;un tube séparé pour déterminer la DO 260 / DO 280 Ratio (voir l&#39;étape 3.1 ci-dessous). Extraits Conserver à -80 ° C. </li></ol> 3. Préparation des extraits de cellules de mammifères <ol><li> Pour les cellules adhérentes, grandir en 100 mm à plat confluent ~ 70%. Vous aurez besoin d&#39;un plat de 100 mm par 11 ml de gradient (rendement typique est d&#39;environ 20 260 unités OD). Échelle du montant linéairement pour des tailles plus grandes ou plus petites gradient. Pour des cellules non adhérentes, soit environ 1 x 10 7 cellules sont nécessaires par 11 ml de gradient. </li><li> Avant de lyse, ajouter cycloheximide à 100 pg / mL. Incuber à 37 ° C pendant 15 min. Transfert des plaques de glace et rincer les cellules à deux reprises en PBS glacé. Toutes les étapes futures doivent être effectuées à 0-4 ° C. </li><li> Enlevez toutes les traces de PBS par aspiration (laissez égoutter les plaques sur un lit d&#39;angle de la glace) et ajouter 1 ml de tampon de lyse, gratter et transfert à l&#39;avant-tube de 1,5 ml refroidi. Composants du tampon de lyse devra être optimisé pour le type de cellule et des conditions de croissance. Un tampon de départ serait de 20 mM HEPES-KOH, pH 7,4, 15 mM MgCl2, 200 mM de KCl, 1% de Triton X-100 (v / v), 100 ug / ml de cycloheximide, 2 mM de DTT, et 1 mg / ml d&#39;héparine. Les principales variables sont les concentrations de magnésium et de chlorure de potassium ainsi que l&#39;inclusion ou l&#39;exclusion de détergents non ioniques afin d&#39;améliorer l&#39;extraction du cytosquelette ou membrane polysomes liés. Lyse des cellules en passant à travers 27 aiguille de la seringue de calibre double ou par coups de 5-10 homogénéisation Dounce de type B en utilisant un pilon. La seringue ou homogénéisateur doit être pré-refroidis avant utilisation. </li><li> Centrifuger à 14000 xg pendant 5 min dans microcentrifugeuse réfrigérés. Transfert lysat frais au gradient de saccharose (pour les cellules de mammifères présente est constitué de tampon de lyse manque de Triton X-100 et l&#39;héparine mais autrement comme décrit dans 1.2 et 1.3) ou geler éclair dans l&#39;azote liquide pour une utilisation ultérieure. </li></ol&gt; 4. La centrifugation de gradients <ol><li> Dégel lysats polyribosomes sur la glace. Déterminer la concentration d&#39;acide nucléique dans le lysat en ajoutant l&#39;échantillon de 10 uL réservés à 1 mL d&#39;eau et de lecture de la DO 260 / DO 280 ratio avec un spectrophotomètre. Rendement typique d&#39;une culture de levures cultivées comme décrit est de 50 unités de DO. </li><li> Avec une pipette avec la pointe placée contre le mur du tube près de la surface de la pente, doucement couche 25 DO 260 unités du lysat sur ​​le haut de la pente. Pour un gradient de 35 500 ml de lysat est habituellement chargé. Tampon de lyse peut être utilisé pour assurer tous les dégradés sont chargés avec un volume égal. Basse quantités de matière de rendement meilleure résolution. En utilisant des pinces, soigneusement bas les gradients dans des seaux d&#39;un rotor swing. </li><li> Centrifuger les gradients à 100.000 xg (23 000 rpm dans un rotor de 630 Surespin) pendant 4 heures à 4 ° C. Ce protocole peut être adapté pour des gradients de plus petit volume. </li></ol> 5. Collecte des données et des fractions <ol><li> Environ 30 min avant la fin de la rotation de 4 heures à fixer l&#39;aiguille le perceur tube modèle 184. Attacher le stylo pour le modèle en ligne Isco UA-5 Absorbance / Monitor fluorescence. Puissance sur l&#39;absorbance suivre pour permettre à une base stable pour être atteint avant l&#39;analyse des échantillons. Électroniques pour l&#39;acquisition du profil de polysomes peut facilement être obtenue en attachant une unité DI-148U acquisition de données (DATAQ Instruments) à l&#39;enregistreur graphique. Les profils peuvent ensuite être collectées en temps réel et sauvegardés pour plus tard en utilisant le logiciel d&#39;annotation WinDaq accompagnement. </li><li> Remplir la pompe seringue avec 50 ml de Fluorinert utilisant le flux inverse, à prise rapide. Assurez-vous qu&#39;il n&#39;ya pas de bulles d&#39;air présentes. Raccordez le tuyau de la seringue pour le perceur tube et mettez brièvement sur les flux de Fluorinert à 3 mL / min dans le sens avant d&#39;effacer le tuyau d&#39;éventuelles bulles d&#39;air. </li><li> Placer un bécher de déchets ou une série de tubes si la collecte de fractions sous le tuyau à l&#39;extrémité de la cellule de flux pour recueillir l&#39;échantillon de ruissellement. </li><li> Retirer délicatement les tubes à centrifuger polyallomère contenant le gradient de saccharose de rotor de la centrifugeuse et les placer sur la glace (pré-former des puits dans la glace avec un tube vide pour éviter de perturber les gradients). </li><li> Pour analyser des extraits de cellules, placer le tube de gradient sur le pierceur tube. Branchez le haut du tube à la sortie et sécurisé. Soulever l&#39;étage inférieur du tube de centrifugation afin que l&#39;aiguille perce le fond du tube. Une fois l&#39;aiguille dépassait de la partie inférieure du tube de centrifugation, amorcer l&#39;écoulement de Fluorinert dans le sens direct au 6mL/min. </li><li> Une fois le Fluorinert a commencé coule dans le tube, de surveiller le mouvement de l&#39;aiguille sur le moniteur d&#39;absorbance en ligne. Une fois que l&#39;aiguille commence à se déplacer, tourner sur le mouvement graphique à un réglage de la vitesse de 60. Le moniteur enregistre la DO 254 en commençant par le matériel gratuitement suivie par la détection sous-unité ribosomale 40S et en continuant par le biais de complexes polyribosomes. Si vous êtes recueillant des fractions d&#39;ARN ou d&#39;analyse des protéines, typiquement 0,2 min fractions sont prises (1 ml). Pour l&#39;analyse de l&#39;ARN, les fractions doivent être recueillis directement dans des tubes contenant Trizol (Invitrogen), le phénol ou SDS / protéinase K, selon la méthode préférée de l&#39;extraction. </li><li> Quand la fin du profil polyribosomes a été atteint, fermer le robinet de Fluorinert le tube de centrifugeuse et d&#39;arrêter le mouvement graphique de l&#39;absorbance / moniteur de fluorescence. </li><li> Rentrez les Fluorinert du tube à centrifuger dans la seringue en commençant par le flux dans le sens inverse à une vitesse rapide en veillant à ne pas tirer de saccharose dans le tube dans la seringue. Dévissez le tube de centrifugation de l&#39;pierceur tube, abaisser l&#39;aiguille, et retirer le tube. </li><li> Répétez les étapes 5.5 à 5.8 pour chaque gradient de saccharose. </li><li> Lorsque tous les gradients de sucrose ont été analysés, rétracter la Fluorinert du tuyau de retour dans la seringue. Retirez chaque partie de fractionnement (perceur de tubes et de cellules d&#39;écoulement), y compris le tuyau de déchets situé en haut et bien rincer avec de l&#39;eau. </li></ol> 6. Les résultats représentatifs <img src="/files/ftp_upload/1948/1948fig1.jpg" alt="Figure 1" /> Figure 1. Oligo-Représentant de l&#39;extrait préparé à partir de ribosome BY4741 souche de levure (tapis une his3 Δ Δ 0 1 leu2 met15 Δ 0 ura3 Δ 0), en présence de cycloheximide. L&#39;extrait a été fractionné en utilisant ribosomique un gradient de saccharose 7 47% et analysées en utilisant un appareil de la seringue de la pompe attachée à un détecteur UV. Peaks contenant polyribosomes et 80S, 60S et 40S des ribosomes sont indiqués.

<ol>
	<li>Baim, S. B., Pietras, D. F., Eustice, D. C., Sherman, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mutation+allowing+an+mRNA+secondary+structure+diminishes+translation+of+Saccharomyces+cerevisiae+iso-1-cytochrome+C.">A mutation allowing an mRNA secondary structure diminishes translation of Saccharomyces cerevisiae iso-1-cytochrome C.</a> Mol. Cell. Biol. 5, 1839-1846 (1985).</li><li>Ripmaster, T. L., Vaughn, G. P., Woolford, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DRS1+to+DRS7,+novel+genes+required+for+ribosome+assembly+and+function+in+Saccharomyces+cerevisiae.">DRS1 to DRS7, novel genes required for ribosome assembly and function in Saccharomyces cerevisiae.</a> Mol. Cell. Biol. 13, 7901-7912 (1993).</li><li>Anand, M., Chakraburtty, K., Marton, M. J., Hinnebusch, A. G., Kinzy, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+interactions+between+yeast+translation+eukaryotic+elongation+factor+(eEF)+1A+and+eEF3.">Functional interactions between yeast translation eukaryotic elongation factor (eEF) 1A and eEF3.</a> J Biol Chem. 278, 6985-6991 (2003).</li><li>Serikawa, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transcriptome+and+its+translation+during+recovery+from+cell+cycle+arrest+in+Saccharomyces+cerevisiae.">The transcriptome and its translation during recovery from cell cycle arrest in Saccharomyces cerevisiae.</a> Mol Cell Proteomics. 2, 191-204 (2003).</li><li>Ingolia, N. T., Ghaemmaghami, S., Newman, J. R., Weissman, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+in+vivo+of+translation+with+nucleotide+resolution+using+ribosome+profiling.">Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling.</a> Science. 324, 218-223 (2009).</li><li>Valasek, L., Szamecz, B., Hinnebusch, A. G., Nielsen, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+stabilization+of+preinitiation+complexes+by+formaldehyde+cross-linking.">In vivo stabilization of preinitiation complexes by formaldehyde cross-linking.</a> Methods Enzymol. 429, 163-183 (2007).</li><li>Fletcher, J. E., Copeland, P. R., Driscoll, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polysome+distribution+of+phospholipid+hydroperoxide+glutathione+peroxidase+mRNA:+evidence+for+a+block+in+elongation+at+the+UGA/selenocysteine+codon.">Polysome distribution of phospholipid hydroperoxide glutathione peroxidase mRNA: evidence for a block in elongation at the UGA/selenocysteine codon.</a> Rna. 6, 1573-1584 (2000).</li><li>Ruan, H. J., Brown, C. Y., Morris, D. R., Richter, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Analysis of mRNA formation and function. , 305-321 (1997).</li><li>Martin, G. W., Berry, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selenocysteine+codons+decrease+polysome+association+on+endogenous+selenoprotein+mRNAs.">Selenocysteine codons decrease polysome association on endogenous selenoprotein mRNAs.</a> Genes Cells. 6, 121-129 (2001).</li><li>Lee, Y. Y., Cevallos, R. C., Jan, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+upstream+open+reading+frame+regulates+translation+of+GADD34+during+cellular+stresses+that+induce+eIF2alpha+phosphorylation.">An upstream open reading frame regulates translation of GADD34 during cellular stresses that induce eIF2alpha phosphorylation.</a> J Biol Chem. 284, 6661-6673 (2009).</li></ol>

Les informations obtenues par le profil de polyribosomes peuvent fournir des informations précieuses sur l&#39;état de translation de la cellule. En outre, le statut de l&#39;assemblage de sous-unités ribosomales eux-mêmes peuvent être déterminés 2. Par exemple la présence de halfmers ou 80S et les grands pics avec un léger épaulement vers la droite sur le profil indique une sous-unité 40S liés attendant 60S sous-unité d&#39;assemblage. Exécution de l&#39;expérience en l&#39;absence de toute cycloheximide ajouté ou autre inhibiteur de l&#39;allongement permet une analyse du taux de ruissellement, ce qui indique si oui ou non l&#39;élongation est modifiée 3. Les fractions sont eux-mêmes une source riche de réactifs pour la détermination ultérieure de l&#39;association d&#39;un ARNm spécifique ou protéine ribosomique avec des sous-populations par Northern blot ou Western des fractions. Le pool total des ARNm associés aux ribosomes actifs peut être déterminé via une puce associée 4 ou 5 profonde analyse par séquençage. Les associations de protéines peuvent également être stabilisés par l&#39;ajout approprié d&#39;une réticulation réactif 6. L&#39;analyse des cellules de mammifère polyribosome convient également de mentionner comme un protocole distinct en termes de difficultés apparentes dans l&#39;établissement des conditions requises pour obtenir polysomes stable. Les systèmes tampons rapportés dans la littérature sont très variables 7-10, donc il peut y avoir une exigence pour un type de cellule optimisation spécifique du tampon de lyse, ou il est tout aussi plausible que le système tampon n&#39;est pas aussi important que l&#39;attention désireux de travailler avec lysats frais conservés à basse température. A notre connaissance une évaluation systématique des paramètres affectant la formation de polysomes mammifères n&#39;a été signalé.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>polyallomer ultracentrifuge tube</td>
<td>&nbsp;</td>
<td>Beckman</td>
<td>326823</td>
<td>1 x 3.5 in. (25x89mm)</td>
</tr>
<tr>
<td>Fluorinert</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>F9755</td>
<td>&nbsp;</td>
<tr>
<td>Cycloheximde</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C-7698</td>
<td>&nbsp;</td>
<tr>
<td>Heparin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H3393</td>
<td>&nbsp;</td>
<tr>
<td>BY4741</td>
<td>&nbsp;</td>
<td>Open Biosystems</td>
<td>YSC1048</td>
<td>Other strains work equally well</td>
</tr>		</tbody>
		</table>
		</div>

eukaryotic polyribosome profile analysis

La synthèse des protéines est un processus complexe cellulaire, qui est réglementé à plusieurs niveaux. Par exemple, la traduction globale peut être inhibé à la phase d&#39;initiation ou de la phase d&#39;élongation par une variété de stress cellulaires tels que la famine ou le retrait des acides aminés du facteur de croissance. Sinon, la traduction des ARNm individuels peuvent être régulées par la localisation d&#39;ARNm ou de la présence de microARN apparenté. Les études de synthèse des protéines utilisent fréquemment l&#39;analyse polyribosome faire la lumière sur les mécanismes de régulation de traduction ou de défauts dans la synthèse des protéines. Dans cet essai, l&#39;ARNm / ribosome complexes sont isolés à partir des cellules eucaryotes. Un gradient de densité de sucrose sépare ARNm liés aux ribosomes multiples connu sous le nom polyribosomes à partir d&#39;ARNm lié à un ribosome simple ou monosome. Fractionnement des gradients permet d&#39;isoler et de quantifier les différentes populations du ribosome et leurs ARNm ou des protéines associées. Les différences dans le rapport de polyribosomes au monosomes dans des conditions définies peuvent être indicatifs de défauts dans l&#39;initiation de traduction ou d&#39;élongation / résiliation. L&#39;examen des ARNm présents dans les fractions polyribosomes peuvent révéler si la cohorte des ARNm individuels en cours de traduction aux modifications des conditions expérimentales. En outre, l&#39;assemblage des ribosomes peut être surveillé par l&#39;analyse des pics de petites et grandes sous-unité ribosomale qui sont aussi séparés par la pente. Dans cette vidéo, nous présentons une méthode pour la préparation des extraits bruts ribosomique de cellules de levure, la séparation de l&#39;extrait par gradient de saccharose et de l&#39;interprétation des résultats. Cette procédure est facilement adaptable aux cellules de mammifères.

Watch this Scientific Journal Video about Eukaryotic Polyribosome Profile Analysis at JoVE.com

Eukaryotic Polyribosome Profile Analysis

Cet article décrit un protocole pour l&#39;extraction de traduire les ribosomes des cellules eucaryotes. Une fois extraites, les ribosomes sont séparés en monosomes et polyribosomes par fractionnement sur gradient de saccharose pour permettre à différentes populations ribosomique pour être analysé. En tant que tel, cette méthode est le gold standard pour examiner la régulation de la traduction.

Eucaryotes Analyse du profil polyribosome

Protein synthesis is a complex cellular process that is regulated at many levels. For example, global translation can be inhibited at the initiation phase ...

Research

JoVE Journal

Biology

52.4K vues.  University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School. Cet article décrit un protocole pour l'extraction de traduire les ribosomes des cellules eucaryotes. Une fois extraites, les ribosomes sont séparés en monosomes et polyribosomes par fractionnement sur gradient de saccharose pour permettre à différentes populations ribosomique pour être analysé. En tant que tel, cette méthode est le gold standard pour examiner la régulation de la traduction.

Vidéo: Eukaryotic Polyribosome Profile Analysis

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

Un neuronale et astrocytes de co-culture de dosage pour l&#39;analyse du contenu haute de la neurotoxicité

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Recherche

Biologie

Bioenergetic Profile Experiment using C2C12 Myoblast Cells

The ability to measure cellular metabolism and understand mitochondrial dysfunction, has enabled scientists worldwide to advance their research in understanding the role of mitochondrial function in obesity, diabetes, aging, cancer, cardiovascular function and safety toxicity.
Cellular metabolism is the process of substrate uptake, such as oxygen, glucose, fatty acids, and glutamine, and subsequent energy conversion through a series of enzymatically controlled oxidation and reduction reactions. These intracellular biochemical reactions result in the production of ATP, the release of heat and chemical byproducts, such as lactate and CO2 into the extracellular environment.
Valuable insight into the physiological state of cells, and the alteration of the state of those cells, can be gained through measuring the rate of oxygen consumed by the cells, an indicator of mitochondrial respiration - the Oxygen Consumption Rate - or OCR. Cells also generate ATP through glycolysis, i.e.: the conversion of glucose to lactate, independent of oxygen. In cultured wells, lactate is the primary source of protons. Measuring the lactic acid produced indirectly via protons released into the extracellular medium surrounding the cells, which causes acidification of the medium provides the Extra-Cellular Acidification Rate - or ECAR.
In this experiment, C2C12 myoblast cells are seeded at a given density in Seahorse cell culture plates. The basal oxygen consumption (OCR) and extracellular acidification (ECAR) rates are measured to establish baseline rates. The cells are then metabolically perturbed by three additions of different compounds (in succession) that shift the bioenergetic profile of the cell.
This assay is derived from a classic experiment to assess mitochondria and serves as a framework with which to build more complex experiments aimed at understanding both physiologic and pathophysiologic function of mitochondria and to predict the ability of cells to respond to stress and/or insults.

A description of a method for profiling mitochondrial function in cells is provided. The mitochondrial profile generated provides four parameters of mitochondrial function that can be measured in one experiment: basal respiration rate, ATP-linked respiration, proton leak, and reserve capacity.

Expérience profil bioénergétique utilisant cellules C2C12 de myoblastes

The ability to measure cellular metabolism and understand mitochondrial dysfunction, has enabled scientists worldwide to advance their research in ...

Correlative Light and Electron Microscopy (CLEM) as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets

The eukaryotic cell relies on complex, highly regulated, and functionally distinct membrane bound compartments that preserve a biochemical polarity necessary for proper cellular function. Understanding how the enzymes, proteins, and cytoskeletal components govern and maintain this biochemical segregation is therefore of paramount importance. The use of fluorescently tagged molecules to localize to and/or perturb subcellular compartments has yielded a wealth of knowledge and advanced our understanding of cellular regulation. Imaging techniques such as fluorescent and confocal microscopy make ascertaining the position of a fluorescently tagged small molecule relatively straightforward, however the resolution of very small structures is limited 1.
On the other hand, electron microscopy has revealed details of subcellular morphology at very high resolution, but its static nature makes it difficult to measure highly dynamic processes with precision 2,3. Thus, the combination of light microscopy with electron microscopy of the same sample, termed Correlative Light and Electron Microscopy (CLEM) 4,5, affords the dual advantages of ultrafast fluorescent imaging with the high-resolution of electron microscopy 6. This powerful technique has been implemented to study many aspects of cell biology 5,7. Since its inception, this procedure has increased our ability to distinguish subcellular architectures and morphologies at high resolution. 
Here, we present a streamlined method for performing rapid microinjection followed by CLEM (Fig. 1). The microinjection CLEM procedure can be used to introduce specific quantities of small molecules and/or proteins directly into the eukaryotic cell cytoplasm and study the effects from millimeter to multi-nanometer resolution (Fig. 2). The technique is based on microinjecting cells grown on laser etched glass gridded coverslips affixed to the bottom of live cell dishes and imaging with both confocal fluorescent and electron microscopy. Localization of the cell(s) of interest is facilitated by the grid pattern, which is easily transferred, along with the cells of interest, to the Epon resin used for immobilization of samples and sectioning prior to electron microscopy analysis (Fig. 3). Overlay of fluorescent and EM images allows the user to determine the subcellular localization as well as any morphological and/or ultrastructural changes induced by the microinjected molecule of interest (Fig. 4). This technique is amenable to time points ranging from &le;5 s up to several hours, depending on the nature of the microinjected sample.

Correlative Light and Electron Microscopy CLEM as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets

The CLEM technique has been adapted to analyze ultrastructural morphology of membranes, organelles, and subcellular structures affected by microinjected molecules. This method combines the powerful techniques of micromanipulation/microinjection, confocal fluorescent microscopy, and electron microscopy to allow millimeter to multi-nanometer resolution. This technique is amenable to a wide variety of applications.

Lumière corrélative et microscopie électronique (CLEM) comme un outil pour visualiser des molécules et de leurs microinjecté eucaryotes Sous-cellulaires cibles


The eukaryotic cell relies on complex, highly regulated, and functionally distinct membrane bound compartments that preserve a biochemical polarity ...

Establishment of Microbial Eukaryotic Enrichment Cultures from a Chemically Stratified Antarctic Lake and Assessment of Carbon Fixation Potential

Lake Bonney is one of numerous permanently ice-covered lakes located in the McMurdo Dry Valleys, Antarctica. The perennial ice cover maintains a chemically stratified water column and unlike other inland bodies of water, largely prevents external input of carbon and nutrients from streams. Biota are exposed to numerous environmental stresses, including year-round severe nutrient deficiency, low temperatures, extreme shade, hypersalinity, and 24-hour darkness during the winter 1. These extreme environmental conditions limit the biota in Lake Bonney almost exclusively to microorganisms 2.
Single-celled microbial eukaryotes (called "protists") are important players in global biogeochemical cycling 3 and play important ecological roles in the cycling of carbon in the dry valley lakes, occupying both primary and tertiary roles in the aquatic food web. In the dry valley aquatic food web, protists that fix inorganic carbon (autotrophy) are the major producers of organic carbon for organotrophic organisms 4, 2. Phagotrophic or heterotrophic protists capable of ingesting bacteria and smaller protists act as the top predators in the food web 5. Last, an unknown proportion of the protist population is capable of combined mixotrophic metabolism 6, 7. Mixotrophy in protists involves the ability to combine photosynthetic capability with phagotrophic ingestion of prey microorganisms. This form of mixotrophy differs from mixotrophic metabolism in bacterial species, which generally involves uptake dissolved carbon molecules. There are currently very few protist isolates from permanently ice-capped polar lakes, and studies of protist diversity and ecology in this extreme environment have been limited 8, 4, 9, 10, 5. A better understanding of protist metabolic versatility in the simple dry valley lake food web will aid in the development of models for the role of protists in the global carbon cycle.
We employed an enrichment culture approach to isolate potentially phototrophic and mixotrophic protists from Lake Bonney. Sampling depths in the water column were chosen based on the location of primary production maxima and protist phylogenetic diversity 4, 11, as well as variability in major abiotic factors affecting protist trophic modes: shallow sampling depths are limited for major nutrients, while deeper sampling depths are limited by light availability. In addition, lake water samples were supplemented with multiple types of growth media to promote the growth of a variety of phototrophic organisms.
RubisCO catalyzes the rate limiting step in the Calvin Benson Bassham (CBB) cycle, the major pathway by which autotrophic organisms fix inorganic carbon and provide organic carbon for higher trophic levels in aquatic and terrestrial food webs 12. In this study, we applied a radioisotope assay modified for filtered samples 13 to monitor maximum carboxylase activity as a proxy for carbon fixation potential and metabolic versatility in the Lake Bonney enrichment cultures.

Microbial eukaryotes are both a source of photosynthetically-derived carbon and top predatory species in permanently ice-covered Antarctic lakes. This report describes an enrichment culture approach to isolate metabolically versatile microbial eukaryotes from the Antarctic lake, Lake Bonney, and assesses inorganic carbon fixation potential using a radioisotope assay for Ribulose-1,5-bisphophate carboxylase oxygenase (RubisCO) activity.

Mise en place de cultures microbiennes eucaryotes à partir d&#39;un enrichissement chimique stratifié lac Antarctique et de l&#39;évaluation du potentiel de fixation du carbone

Lake Bonney is one of numerous permanently ice-covered lakes located in the McMurdo Dry Valleys, Antarctica. The perennial ice cover maintains a ...

Metabolic Profile Analysis of Zebrafish Embryos

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these relationships will help to understand the underlying etiology for a range of diseases linked with mitochondrial dysfunction, such as diabetes and obesity. Recent advances in instrumentation, has enabled the monitoring of distinct parameters of mitochondrial function in cell lines or tissue explants. Here we present a method for a rapid and sensitive analysis of mitochondrial function parameters in vivo during zebrafish embryonic development using the Seahorse bioscience XF 24 extracellular flux analyser. This protocol utilizes the Islet Capture microplates where a single embryo is placed in each well, allowing measurement of bioenergetics, including: (i) basal respiration; (ii) basal mitochondrial respiration (iii) mitochondrial respiration due to ATP turnover; (iv) mitochondrial uncoupled respiration or proton leak and (iv) maximum respiration. Using this approach embryonic zebrafish respiration parameters can be compared between wild type and genetically altered embryos (mutant, gene over-expression or gene knockdown) or those manipulated pharmacologically. It is anticipated that dissemination of this protocol will provide researchers with new tools to analyse the genetic basis of metabolic disorders in vivo in this relevant vertebrate animal model.

Zebrafish represent a powerful vertebrate model that has been under-utilised for metabolic studies. Here we describe a rapid way to measure the in vivo metabolic profile of developing zebrafish that allows the comparison of different mitochondrial function parameters between genetically or pharmacologically manipulated embryos, thereby increasing the applicability of this organism.

Analyse du profil métabolique des embryons de poisson zèbre

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these ...

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and composed of mature enterocytes, goblet cells and enteroendocrine cells; and crypts, residing proximal to the submucosa and the muscularis, harboring adult stem and progenitor cells and mature Paneth cells, as well as stromal and immune cells of the crypt microenvironment. Until the last few years, in vitro studies of small intestine was limited to cell lines derived from either benign or malignant tumors, and did not represent the physiology of normal intestinal epithelia and the influence of the microenvironment in which they reside. Here, we demonstrate a method adapted from Sato et al. (2009) for culturing primary mouse intestinal crypt organoids derived from C57BL/6 mice. In addition, we present the use of crypt organoid cultures to assay the crypt metabolic profile in real time by measurement of basal oxygen consumption, glycolytic rate, ATP production and respiratory capacity. Organoids maintain properties defined by their source and retain aspects of their metabolic adaptation reflected by oxygen consumption and extracellular acidification rates. Real time metabolic studies in this crypt organoid culture system are a powerful tool to study crypt organoid energy metabolism, and how it can be modulated by nutritional and pharmacological factors.

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

Small intestinal crypt organoids cultured ex vivo provide a tissue culture system that recapitulates growth of crypts dependent on stem cells and their niche. We established a method to assay the metabolic profile in real time in primary mouse crypt organoids. We found organoids maintain physiological properties defined by their source.

Analyse en temps réel de profil métabolique dans<em&gt; Ex Vivo</em&gt; Cultures souris intestinale Crypte organoïde

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and ...

Genome-wide Purification of Extrachromosomal Circular DNA from Eukaryotic Cells

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs contribute to genetic variation among somatic cells in multicellular organisms and to evolution of unicellular eukaryotes. Sensitive methods for detecting eccDNA are needed to clarify how these elements affect genome stability and how environmental and biological factors&#160;induce their formation in eukaryotic cells. This video presents a sensitive eccDNA-purification method called Circle-Seq. The method encompasses column purification of circular DNA, removal of remaining linear chromosomal DNA, rolling-circle amplification of eccDNA, deep sequencing, and mapping. Extensive exonuclease treatment&#160;was&#160;required for sufficient linear chromosomal DNA degradation. The rolling-circle amplification step by &#966;29 polymerase enriched for circular DNA over linear DNA. Validation of the Circle-Seq method on three S. cerevisiae CEN.PK populations of 1010 cells detected hundreds of eccDNA profiles in sizes larger than 1 kilobase. Repeated findings of ASP3-1, COS111, CUP1, RSC30,&#160;HXT6, HXT7 genes on circular DNA in both S288c and CEN.PK suggests that DNA circularization is conserved between strains at these loci. In sum, the Circle-Seq method has broad applicability for genome-scale screening for eccDNA in eukaryotes as well as for detecting specific eccDNA types.

This paper presents a sensitive method called Circle-Seq for purifying extrachromosomal circular DNA (eccDNA). The method encompasses column purification, removal of remaining linear chromosomal DNA, rolling-circle amplification and high-throughput sequencing. Circle-Seq is applicable to genome-scale screening of eukaryotic eccDNA and studying genome instability and copy-number variation.

Purification de l&#39;ensemble du génome de l&#39;ADN extrachromosomique circulaire à partir de cellules eucaryotes

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs ...

Laboratory Protocol for Genetic Gut Content Analyses of Aquatic Macroinvertebrates Using Group-specific rDNA Primers

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the invasion of non-indigenous species. However, an exact identification of field predator-prey interactions is difficult in many cases. These analyses are often based on a visual evaluation of gut content or the analysis of stable isotope ratios (&#948;15N and &#948;13C). Such methods require comprehensive knowledge about, respectively, morphologic diversity or isotopic signature from individual prey organisms, leading to obstacles in the exact identification of prey organisms. Visual gut content analyses especially underestimate soft bodied prey organisms, because maceration, ingestion and digestion of prey organisms make identification of specific species difficult. Hence, polymerase chain reaction (PCR) based strategies, for example the use of group-specific primer sets, provide a powerful tool for the investigation of food web interactions. Here, we describe detailed protocols to investigate the gut contents of macroinvertebrate consumers from the field using group-specific primer sets for nuclear ribosomal deoxyribonucleic acid (rDNA). DNA can be extracted either from whole specimens (in the case of small taxa) or out of gut contents of specimens collected in the field. Presence and functional efficiency of the DNA templates need to be confirmed directly from the tested individual using universal primer sets targeting the respective subunit of DNA. We also demonstrate that consumed prey can be determined further down to species level via PCR with unmodified group-specific primers combined with subsequent single strand conformation polymorphism (SSCP) analyses using polyacrylamide gels. Furthermore, we show that the use of different fluorescent dyes as labels enables parallel screening for DNA fragments of different prey groups from multiple gut content samples via automated fragment analysis.

Most common gut content analyses of macroinvertebrates are visual. Requiring intense knowledge about morphological diversity of prey organisms, they miss soft bodied prey and, due to strong comminution of prey, are nearly impossible for some organisms, including amphipods. We provide detailed, novel genetic approaches for macroinvertebrate prey identification in the diet of amphipods.

Protocole de laboratoire pour les Analyses de contenu Gut génétiques de macroinvertébrés aquatiques Using Group-specific ADNr amorces

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the ...

Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy (SEM)

Scanning electron microscopy (SEM) is a widely available technique that has been applied to study biological specimens ranging from individual proteins to cells, tissues, organelles, and even whole organisms. This protocol focuses on two chemical drying methods, hexamethyldisilazane (HMDS) and t-butyl alcohol (TBA), and their application to imaging of both prokaryotic and eukaryotic organisms using SEM. In this article, we describe how to fix, wash, dehydrate, dry, mount, sputter coat, and image three types of organisms: cyanobacteria (Toxifilum mysidocida, Golenkina sp., and an unknown sp.), two euglenoids from the genus Monomorphina (M. aenigmatica and M. pseudopyrum), and the fruit fly (Drosophila melanogaster). The purpose of this protocol is to describe a fast, inexpensive, and simple method to obtain detailed information about the structure, size, and surface characteristics of specimens that can be broadly applied to a large range of organisms for morphological assessment. Successful completion of this protocol will allow others to use SEM to visualize samples by applying these techniques to their system.

Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy SEM

SEM analysis is an effective method to aid in species identification or phenotypic discrimination. This protocol describes the methods for examining specific morphological details of three representative types of organisms and would be broadly applicable to examining features of many organismal and tissue types.

Préparation des organismes procaryotes et eucaryotes à l’aide de séchage chimique pour l’analyse morphologique en microscopie électronique (MEB)

Scanning electron microscopy (SEM) is a widely available technique that has been applied to study biological specimens ranging from individual proteins to ...

Image Rendering Techniques in Postmortem Computed Tomography: Evaluation of Biological Health and Profile in Stranded Cetaceans

With 6 years of experience in implementing virtopsy routinely into the Hong Kong cetacean stranding response program, standardized virtopsy procedures, postmortem computed tomography (PMCT) acquisition, postprocessing, and evaluation were successfully established. In this pioneer cetacean virtopsy stranding response program, PMCT was performed on 193 stranded cetaceans, providing postmortem findings to aid necropsy and shed light on the biological health and profile of the animals. This study aimed to assess 8 image rendering techniques in PMCT, including multiplanar reconstruction, curved planar reformation, maximum intensity projection, minimum intensity projection, direct volume rendering, segmentation, transfer function, and perspective volume rendering. Illustrated with practical examples, these techniques were able to identify most of the PM findings in stranded cetaceans and served as a tool to investigate their biological health and profile. This study could guide radiologists, clinicians and veterinarians through the often difficult and complicated realm of PMCT image rendering and reviewing.

The Hong Kong cetacean stranding response program has incorporated postmortem computed tomography, which provides valuable information on the biological health and profile of the deceased animals. This study describes 8 image rendering techniques that are essential for the identification and visualization of postmortem findings in stranded cetaceans, which will help clinicians, veterinarians and stranding response personnel worldwide to fully utilize the radiological modality.

Techniques de rendu d’image dans la tomographie calculée post mortem : Évaluation de la santé et du profil biologiques chez les cétacés échoués

With 6 years of experience in implementing virtopsy routinely into the Hong Kong cetacean stranding response program, standardized virtopsy procedures, ...