Ces efforts de recherche ont été soutenus par la Division de la National Science Foundation de la chimie, bio-ingénierie, l&#39;environnement, et les transports (CBET) Systèmes sous les numéros d&#39;attribution ÉFAC-0853780 et ÉFAC-1159344 et le National Institutes of Health, National Institute of Environmental Health Sciences (NIEHS) sous sentence nombre 1R43ES022567-01, et l&#39;Institut national du cancer, programme d&#39;imagerie du cancer sous récompense numéro CA127745-01. L&#39;instrument Lumina SIIV utilisée dans ce travail a été obtenue à partir de l&#39;Armée de Défense Programme d&#39;Instrumentation de recherche de l&#39;Université américaine.

1. Préparation des cellules <ol><li> Récupérez un flacon de cellules bioluminescentes HEK293 de l&#39;azote stock congelé liquide et les cultiver dans un milieu de Eagle modifié par Dulbecco (DMEM) supplémenté avec 10% de sérum de veau foetal, 0,01 mm acides aminés non essentiels, 1x antibiotiques antimycosiques et 0,01 mM pyruvate de sodium dans un T 75 flacon de culture tissulaire à 37 ° C et 5% de CO 2. Remarque: Les médias et les composants supplémentaires varient en fonction de lignées cellulaires et doivent donc être choisis en conséquence. </li><li> Actualiser moyenne tous les 2-3 jours jusqu&#39;à atteindre ~ 80% de confluence. Note: La quantité de cellules nécessaires sera basé sur la conception expérimentale. Si nécessaire, d&#39;autres cellules peuvent être obtenues par repiquage. </li><li> Récolte des cellules pour les tests. <ol><li> Retirez le support et laver les cellules avec du phosphate salin (PBS) tamponné en remuant doucement le ballon et ensuite jeter le passé PBS. </li><li> Ajouter la trypsine et incuber à 37 ° C pendant 2 min,ou jusqu&#39;à ce que les cellules sont détachées de la fiole. </li><li> Recueillir les cellules individuelles dans PBS et les transférer dans un tube à centrifuger propre. Remarque: Si les lignées cellulaires non adhérentes sont utilisés, des cellules séparées par centrifugation et les laver une fois avec PBS. </li></ol></li><li> Déterminer le nombre total de cellules en utilisant un hématimètre ou tout autre système de comptage cellulaire. </li><li> Centrifuger la suspension cellulaire à 300 g pendant 5 min. Jeter le surnageant et remettre en suspension les cellules dans l&#39;eau douce, moyen préchauffé. </li><li> un nombre égal de graines de cellules dans des puits individuels d&#39;une plaque multi-puits opaque. Dans cet exemple, la plaque 5 x 10 5 cellules dans un volume de 1 ml dans chaque puits d&#39;une plaque de 24 puits noir. Plate un volume égal de milieu dans les puits en triple pour agir comme contrôle négatif. Note: Le nombre de cellules étalées dans chaque puits est flexible et peut être optimisée pour des expériences individuelles. Pour chaque ligne de cellules de bioluminescence, de déterminer expérimentalement la relation entre le nombre de cellules et biolumiNESCent sortie, ainsi que la cellule minimale détectable comptent avant tout test de toxicité. Pour ce faire, mesurer la bioluminescence à travers un large éventail de tailles de population (par exemple, 1 x 10 3 à 1 x 10 6 cellules) dans des conditions d&#39;imagerie standardisés. </li><li> Traiter les cellules avec un composé de test (s) tel que décrit ci-dessous. </li></ol> 2. Préparation chimique <ol><li> Préparer la substance chimique testée comme une solution stock concentrée. Dans cet exemple, utiliser un 100 mg / ml de solution mère d&#39;un antibiotique de la famille de la bléomycine. Note: Afin de minimiser les effets toxiques potentiels basés véhicule, surtout si un solvant organique est utilisé comme un véhicule pour l&#39;addition de produits chimiques, il est recommandé que la concentration de l&#39;action soit au moins 1000 fois la concentration post-application souhaitée. </li><li> Ajouter le bouillon chimique préparé directement dans les cellules. Dans cet exemple, la dose de l&#39;antibiotique d&#39;intérêt à des concentrations finales de 100, 200, 300 et 400 pg / ml dans triplicate puits. Laisser trois puits de cellules non traitées comme témoins. Note: La concentration finale de la substance chimique cible doit être choisi en fonction des objectifs expérimentaux spécifiques. Une large gamme de concentrations est normalement testé pour obtenir un éventail complet des effets toxiques. </li></ol> 3. Imagerie et analyse des données <ol><li> Immédiatement après l&#39;addition de produits chimiques, de placer la plaque dans la chambre de formation d&#39;image de l &#39;instrument approprié pour l&#39;acquisition d&#39;images et la mesure de la bioluminescence. </li><li> bioluminescence de mesure toutes les 15 minutes sur une période de 24 heures avec un temps d&#39;intégration de 10 min pour chaque lecture. Note: Le temps d&#39;intégration utilisé dans cet exemple est une base par plaque et peut être réglé pour la mesure sur une base par puits en utilisant d&#39;autres luminomètres de choix. Le temps d&#39;intégration peut également être ajustée en fonction de la lignée cellulaire bioluminescente choisie. Parce que la bioluminescence est produite de manière autonome, sans destruction cellulaire ou toute stimulation externe, lisezréunions peuvent être prises à tout point dans le temps désiré pour atteindre les objectifs expérimentaux spécifiques. </li><li> Quantifier l&#39;intensité de bioluminescence à partir de chaque puits par identification d&#39;une région d&#39;intérêt en utilisant un logiciel compatible. Afficher la sortie de lumière soit en tant que le flux total (photons / seconde), soit la luminance moyenne (photons / seconde / cm 2 / steridian) de chaque échantillon. </li></ol>

<ol>
	<li>U.S. Food and Drug Administration. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+it+really+FDA+approved?.">Is it really FDA approved?.</a> FDA Consumer Health Information. 2, (2009).</li><li>Hartung, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxicology+for+the+twenty-first+century.">Toxicology for the twenty-first century.</a> Nature. 460, 208-212 (2009).</li><li>Olson, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concordance+of+the+toxicity+of+pharmaceuticals+in+humans+and+in+animals.">Concordance of the toxicity of pharmaceuticals in humans and in animals.</a> Regul. Toxicol. Pharmacol. 32, 56-67 (2000).</li><li>Stacey, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+developments+in+cell+culture+technology.">Current developments in cell culture technology.</a> Adv. Exp. Med. Biol. 745, 1-13 (2012).</li><li>Li, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+for+human+ADME/Tox+drug+properties+in+drug+discovery.">Screening for human ADME/Tox drug properties in drug discovery.</a> Drug Discov. Today. 6, 357-366 (2001).</li><li>Li, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+approaches+to+evaluate+ADMET+drug+properties.">In vitro approaches to evaluate ADMET drug properties.</a> Curr. Top. Med. Chem. 4, 701-706 (2004).</li><li>Tannous, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gaussia+luciferase+reporter+assay+for+monitoring+biological+processes+in+culture+and+in+vivo.">Gaussia luciferase reporter assay for monitoring biological processes in culture and in vivo.</a> Nat. Protoc. 4, 582-591 (2009).</li><li>Thompson, E. M., Nagata, S., Tsuji, F. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+and+expression+of+cDNA+for+the+luciferase+from+the+marine+ostracod+Vargula+hilgendorfii.">Cloning and expression of cDNA for the luciferase from the marine ostracod Vargula hilgendorfii.</a> Proc. Natl. Acad. Sci. U.S.A. 86, 6567-6571 (1989).</li><li>Lupold, S. E., Johnson, T., Chowdhury, W. H., Rodriguez, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+real+time+Metridia+luciferase+based+non-invasive+reporter+assay+of+mammalian+cell+viability+and+cytotoxicity+via+the+beta-actin+promoter+and+enhancer.">A real time Metridia luciferase based non-invasive reporter assay of mammalian cell viability and cytotoxicity via the beta-actin promoter and enhancer.</a> PLoS ONE. 7, (2012).</li><li>Meighen, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+biology+of+bacterial+bioluminescence.">Molecular biology of bacterial bioluminescence.</a> Microbiol. Rev. 55, 123-142 (1991).</li><li>Close, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autonomous+bioluminescent+expression+of+the+bacterial+luciferase+gene+cassette+(lux)+in+a+mammalian+cell+line.">Autonomous bioluminescent expression of the bacterial luciferase gene cassette (lux) in a mammalian cell line.</a> PLoS ONE. 5, e12441 (2010).</li><li>Close, D. M., Hahn, R. E., Ripp, S. A., Sayler, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determining+toxicant+bioavailability+using+a+constitutively+bioluminescent+human+cell+line.">Determining toxicant bioavailability using a constitutively bioluminescent human cell line.</a> , 1-4 (2011).</li><li>Oliva-Trastoy, M., Defais, M., Larminat, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resistance+to+the+antibiotic+Zeocin+by+stable+expression+of+the+Sh+ble+gene+does+not+fully+suppress+Zeocin-induced+DNA+cleavage+in+human+cells.">Resistance to the antibiotic Zeocin by stable expression of the Sh ble gene does not fully suppress Zeocin-induced DNA cleavage in human cells.</a> Mutagenesis. 20, 111-114 (2005).</li><li>Close, D. M., Webb, J. D., Ripp, S. A., Patterson, S. S., Sayler, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remote+detection+of+human+toxicants+in+real+time+using+a+human+optimized+bioluminescent+bacterial+luciferase+gene+cassette+bioreporter.">Remote detection of human toxicants in real time using a human optimized bioluminescent bacterial luciferase gene cassette bioreporter.</a> Proc. SPIE 8371, Sensing Technologies for Global Health, Military Medicine, Disaster Response, and Environmental Monitoring II; and Biometric Technology for Human Identification IX. 837117, (2012).</li><li>Close, D. M., Hahn, R. E., Patterson, S. S., Ripp, S. A., Sayler, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+human+optimized+bacterial+luciferase,+firefly+luciferase,+and+green+fluorescent+protein+for+continuous+imaging+of+cell+culture+and+animal+models.">Comparison of human optimized bacterial luciferase, firefly luciferase, and green fluorescent protein for continuous imaging of cell culture and animal models.</a> J. Biomed. Opt. 16, e12441 (2011).</li></ol>

DM Close, SA Ripp et GS Sayler sont les fondateurs et propriétaires de 490 Biotech, Inc.

Cette méthode illustre l&#39;utilisation de cellules de mammifères autonome bioluminescentes comme un test de dépistage de la cytotoxicité in vitro qui permet aux cellules vivantes à être surveillés en permanence au cours de leur vie. Ce protocole est flexible et peut être modifié pour tenir compte des conditions expérimentales spécifiques selon les besoins. Par exemple, l&#39;expérience présentée ici est approprié pour le suivi des effets toxiques aigus, mais peut être adapté pour év...

Aux États-Unis, les produits pharmaceutiques et autres produits destinés à la consommation humaine nécessitent une évaluation approfondie avant qu&#39;ils soient approuvés pour utilisation par les consommateurs par la Food and Drug Administration. Le fardeau financier pour réaliser ce test est placé sur le développeur 1, ce qui augmente considérablement le coût de développement de nouveaux composés et donc se traduit par une augmentation des coûts pour les consommateurs. Alors que traditionnellement une grande partie de ce dépistage a utilisé des sujets animaux pour agir comme mandataires pour des hôtes humains, ce qui s&#39;est avéré être un grand fardeau financier, avec un montant estimé à 2,8 milliards de dollars dépensés chaque année sur ADME / Tox (adsorption, distribution, métabolisme, excrétion et toxicité ) le dépistage seul 2 et preuves croissantes suggérant que les modèles animaux ne peuvent pas prédire de façon fiable les réponses toxicologiques humains 3. Par conséquent, dans les tests de cellules humaines in vitro basée sur la culture a gagné en popularité au cours des deux dernières décennies en raison de sa relativement faiblecoût, un débit plus élevé et une meilleure représentation de la biodisponibilité de l&#39;homme et de la toxicologie 4. Méthodes de dépistage de toxicité culture à base de cellules actuelles emploient divers paramètres, tels que la mesure des niveaux d&#39;ATP, le dépistage de l&#39;activité des enzymes cytoplasmiques endogène disponibles, pour sonder l&#39;intégrité de la membrane cellulaire, ou suivre le niveau de l&#39;activité mitochondriale, pour évaluer la viabilité cellulaire 5 , 6. Cependant, quel que soit le critère choisi, ces méthodes nécessitent tous la destruction de l&#39;échantillon avant que les mesures peuvent être prises, ainsi que la production de données à un seul moment. En conséquence, un grand nombre d&#39;échantillons doivent être préparés et traités en parallèle des études de base de la cinétique toxicologiques, ajoutant encore du coût et de la main-d&#39;œuvre nécessaire pour le développement de nouveaux composés. Alternativement, des tests utilisant sécrétées luciférase comme Gaussia luciférase 7, Vargula luciférase 8 et 9 Metridia luciféraseont été développés qui éliminent la nécessité de lyse cellulaire et nécessitent une fraction des médias pour la mesure du point de terminaison, cependant, ceux-ci sont encore limitées à l&#39;échantillonnage à des moments prédéterminés et nécessite également l&#39;ajout de substrats exogènes lumière d&#39;activation. Pour éviter, au détriment de la destruction de l&#39;échantillon requise ainsi que d&#39;éliminer le coût de substrats, d&#39;une lignée cellulaire humaine a été conçu, qui exprime la bioluminescence bactérienne plein (lux) de la cassette de gène (luxCDABEfrp) pour permettre un suivi continu de cellules vivantes qui est semblable à l&#39;imagerie des cellules vivantes fluorescente à base de colorant, mais sans que les procédures d&#39;enquête photon d&#39;activation et microscopiques supplémentaires. Cette lignée cellulaire est capable de produire de façon constitutive d&#39;un signal optique pour la détection directe en continu sans avoir besoin de stimulation externe, évitant ainsi la destruction de l&#39;échantillon. Mécaniquement, le signal de bioluminescence produite à partir de ces cellules résultes lorsque l&#39;enzyme luciférase luxAB formé catalyse l&#39;oxydation d&#39;un aldéhyde gras à longue chaîne (synthétisé et régénérée par les produits des gènes luxCDE utilisant des substrats endogènes) en présence d&#39;une réduction de phosphate de riboflavine (FMNH2, qui est recyclé à partir de FMN par le gène FRP produit) et de l&#39;oxygène moléculaire 10. L&#39;expression de la cassette lux dans la cellule-hôte permet donc de la lumière à être produit et détecté sans destruction cellulaire ou une addition de substrat exogène. De même, l&#39;interaction entre les gènes lux et la FMN disponible endogène, et O 2 co-substrats, et l&#39;exigence de maintien d&#39;un environnement qui peut soutenir la conversion des FMN à FMNH2, assure que le signal de bioluminescence qui en résulte ne peut être détectée de vivre , les cellules métaboliquement actives. Ces exigences ont déjà été exploités pour démontrer que lux-Based sortie bioluminescence est fortement corrélée avec la taille de la population cellulaire 11 et que l&#39;exposition au composé toxique altère production autobioluminescent de façon dose-réponse 12. Ici, nous utilisons un rein embryonnaire humain autobioluminescent précédemment caractérisé (HEK293) de ligne de la cellule 11 de démontrer le dépistage toxicologique automatisé d&#39;un antibiotique de la famille de la bléomycine avec activité endommageant l&#39;ADN connu comme un exemple représentatif pour valider la demande de cellules de mammifères autobioluminescent pour les tests de toxicité.

<table border="1">
	<tbody>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comment</td>
		</tr>
		<tr>
			<td>IVIS Lumina</td>
			<td>PerkinElmer</td>
			<td></td>
			<td>Other IVIS models and PMT-based plate readers can also be used</td>
		</tr>
		<tr>
			<td>Living Imaging 2.0</td>
			<td>PerkinElmer</td>
			<td></td>
			<td>Newer updates of this software is available</td>
		</tr>
		<tr>
			<td>75 cm2 cell culture treated flasks</td>
			<td>Corning</td>
			<td>430641</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well tissue culture-treated plates</td>
			<td>Costar</td>
			<td>07-200-83</td>
			<td></td>
		</tr>
		<tr>
			<td>Black 24-well plate</td>
			<td>Greiner Bio-One</td>
			<td>662174</td>
			<td>96- or 384-well plates can be used for higher throughput applications</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Hyclone</td>
			<td>SH30910</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM), phenol red-free</td>
			<td>Hyclone</td>
			<td>SH30284</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Hyclone</td>
			<td>SH3091003</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonessential amino acids, 100x</td>
			<td>Life Technologies</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-antimycotic, 100x</td>
			<td>Life Technologies</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate, 100mM</td>
			<td>Life Technologies</td>
			<td>11360070</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeocin, 100 mg/ml</td>
			<td>Life Technologies</td>
			<td>R25001</td>
			<td></td>
		</tr>
		<tr>
			<td>Tripsin, 0.05%</td>
			<td>Life Technologies</td>
			<td>25300062</td>
			<td></td>
		</tr>
	</tbody>
</table>

autonomously bioluminescent mammalian cells for continuous and real-time monitoring of cytotoxicity

Basés sur les cellules de mammifères dans des essais in vitro ont été largement utilisés comme alternatives à l&#39;expérimentation animale pour les études toxicologiques mais ont été limités en raison des coûts financiers et temporels élevés de la préparation d&#39;échantillons en parallèle qui sont rendues nécessaires en raison de la nature destructrice des méthodes de dépistage luciférase à base de luciole . Cette vidéo décrit l&#39;utilisation de cellules de mammifères autonome bioluminescentes, qui ne nécessite pas l&#39;ajout destructrice d&#39;un substrat luciférine, comme une méthode peu coûteuse et facile de surveiller les effets cytotoxiques d&#39;un composé d&#39;intérêt. Cellules de mammifère exprimant de manière stable la bioluminescence bactérienne plein (luxCDABEfrp) de la cassette du gène de produire de manière autonome un signal optique que des pics à 490 nm, sans l&#39;addition d&#39;un substrat de luciférine coûteux et susceptibles d&#39;interférer, l&#39;excitation par une source d&#39;énergie externe, ou la destruction de l&#39;échantillon qui est traditionnellement effectué au cours de la procédure d&#39;imagerie optiques. Cette indépendance de la stimulation extérieure place la charge pour maintenir la réaction de bioluminescence uniquement de la cellule, ce qui signifie que le signal qui en résulte est détectée seulement au cours du métabolisme actif. Cette caractéristique rend la lignée cellulaire lux exprimant un excellent candidat pour une utilisation comme biosentinel contre les effets cytotoxiques parce que les changements dans la production de bioluminescence sont révélateurs d&#39;effets négatifs sur la croissance cellulaire et du métabolisme. De même, le caractère autonome et le manque de destruction de l&#39;échantillon requis permet l&#39;imagerie répétée du même échantillon en temps réel tout au long de la période d&#39;exposition aux substances toxiques et peuvent être effectuées sur plusieurs échantillons en utilisant l&#39;équipement d&#39;imagerie existant de manière automatisée.

Dans cette étude, la dynamique des cellules autobioluminescent HEK293 ont été surveillés en continu sur une période de 24 heures en réponse à l&#39;exposition aux antibiotiques (Figure 1). Les effets toxiques de cet antibiotique, qui est un membre de la famille de la bléomycine connus pour tuer les cellules vivantes en se liant à et cliver l&#39;ADN 13, ont été démontrés par l&#39;intermédiaire d&#39;une diminution de la production de bioluminescence par rapport aux cellules non...

Watch this Scientific Journal Video about Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity at JoVE.com

Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity

Les cellules de mammifères exprimant la cassette du gène de bioluminescence bactérienne (<em&gt; Lux</em&gt;) Produire de la lumière de manière autonome. La dynamique bioluminescentes résultant lors de l&#39;exposition chimique a été démontrée pour tenir compte des effets du traitement sur la croissance cellulaire et le métabolisme, ce qui rend ces cellules un, outil peu coûteux en temps réel continue dépistage toxicité qui peut être facilement adaptée pour l&#39;automatisation à haut débit.

Cellules de mammifères autonome bioluminescentes pour la surveillance continue et en temps réel de la cytotoxicité

Mammalian  cell-based in vitro assays have been  widely employed as alternatives to animal testing for toxicological studies but  have been limited due to ...

autonomously-bioluminescent-mammalian-cells-for-continuous-real-time

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JoVE Journal

Biology

10.0K vues.  Oak Ridge National Laboratory. Les cellules de mammifères exprimant la cassette du gène de bioluminescence bactérienne ( Lux) Produire de la lumière de manière autonome. La dynamique bioluminescentes résultant lors de l'exposition chimique a été démontrée pour tenir compte des effets du traitement sur la croissance cellulaire et le métabolisme, ce qui rend ces cellules un, outil peu coûteux en temps réel continue dépistage toxicité qui peut être facilement adaptée po...

Vidéo: Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity

Trypsinizing and Subculturing Mammalian Cells

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in cell death. The first step in subculturing is to detach cells from the surface of the primary culture vessel by trypsinization or mechanical means. The resultant cell suspension is then subdivided, or reseeded, into fresh cultures. Secondary cultures are checked for growth and fed periodically, and may be subsequently subcultured to produce tertiary cultures. The time between passaging of cells varies with the cell line and depends on the growth rate.

As cells reach confluency, they must be subcultured or passaged. This video will demonstrate a procedure for subculturing both adherent and suspension cells.

Trypsinisation et Repiquage des cellules de mammifères

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in ...

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Biologie

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

EsiRNA MISSION pour le dépistage de l&#39;ARNi dans les cellules de mammifères

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

Mutagenèse et analyse fonctionnelle des canaux ioniques hétérologue exprimée dans les cellules de mammifères

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR (qPCR) Arrays

Quantitative real-time PCR (QPCR) has emerged as an accurate and valuable tool in profiling gene expression levels.  One of its many advantages is a lower detection limit compared to other methods of gene expression profiling while using smaller amounts of input for each assay.  Automated qPCR setup has improved this field by allowing for greater reproducibility.  Its convenient and rapid setup allows for high-throughput experiments, enabling the profiling of many different genes simultaneously in each experiment.  This method along with internal plate controls also reduces experimental variables common to other techniques.  
We recently developed a qPCR assay for profiling of pre-microRNAs  (pre-miRNAs) using a set of 186 primer pairs.  MicroRNAs have emerged as a novel class of small, non-coding RNAs with the ability to regulate many mRNA targets at the post-transcriptional level.  These small RNAs are first transcribed by RNA polymerase II as a primary miRNA (pri-miRNA) transcript, which is then cleaved into the precursor miRNA (pre-miRNA).  Pre-miRNAs are exported to the cytoplasm where Dicer cleaves the hairpin loop to yield mature miRNAs.  Increases in miRNA levels can be observed at both the precursor and mature miRNA levels and profiling of both of these forms can be useful.  There are several commercially available assays for mature miRNAs; however, their high cost may deter researchers from this profiling technique.  Here, we discuss a cost-effective, reliable, SYBR-based qPCR method of profiling pre-miRNAs.  Changes in pre-miRNA levels often reflect mature miRNA changes and can be a useful indicator of mature miRNA expression.  However, simultaneous profiling of both pre-miRNAs and mature miRNAs may be optimal as they can contribute nonredundant information and provide insight into microRNA processing.  Furthermore, the technique described here can be expanded to encompass the profiling of other library sets for specific pathways or pathogens.

Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR qPCR Arrays

We will demonstrate the setup and analysis of pre-microRNA 96-well arrays for QPCR using a robot as well as by hand with a Thermo Scientific Matrix multichannel pipette.

Profilage de la pré-micro ARNs et micro-ARN en utilisant quantitative PCR en temps réel (qPCR) Arrays

Quantitative real-time PCR (QPCR) has emerged as an accurate and valuable tool in profiling gene expression levels.  One of its many advantages is a lower ...

Real-time Monitoring of Ligand-receptor Interactions with Fluorescence Resonance Energy Transfer

FRET is a process whereby energy is non-radiatively transferred from an excited donor molecule to a ground-state acceptor molecule through long-range dipole-dipole interactions1. In the present sensing assay, we utilize an interesting property of PDA: blue-shift in the UV-Vis electronic absorption spectrum of PDA (Figure 1) after an analyte interacts with receptors attached to PDA2,3,4,7. This shift in the PDA absorption spectrum provides changes in the spectral overlap (J) between PDA (acceptor) and rhodamine (donor) that leads to changes in the FRET efficiency. Thus, the interactions between analyte (ligand) and receptors are detected through FRET between donor fluorophores and PDA. In particular, we show the sensing of a model protein molecule streptavidin. We also demonstrate the covalent-binding of bovine serum albumin (BSA) to the liposome surface with FRET mechanism. These interactions between the bilayer liposomes and protein molecules can be sensed in real-time. The proposed method is a general method for sensing small chemical and large biochemical molecules. Since fluorescence is intrinsically more sensitive than colorimetry, the detection limit of the assay can be in sub-nanomolar range or lower8. Further, PDA can act as a universal acceptor in FRET, which means that multiple sensors can be developed with PDA (acceptor) functionalized with donors and different receptors attached on the surface of PDA liposomes.

We demonstrate FRET between conjugated polymer polydiacetylene (PDA) and fluorophore attached to the surface of PDA liposomes for the sensing of biomolecules. PDA liposomes also contained receptor molecules on their surfaces for biomolecules to be used as probes. Ligand-receptor interactions lead to changes in the FRET efficiency between the fluorophore and PDA which is the basis of the sensing mechanism.

Surveillance en temps réel des interactions ligand-récepteur avec Fluorescence Resonance Energy Transfer

FRET is a process  whereby energy is non-radiatively transferred from an excited donor molecule to  a ground-state acceptor molecule through long-range ...

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

F&ouml;rster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo1-2. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest3-4. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells5, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium6, cAMP7-8, inositol phosphates9 and cGMP10-11. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a &beta;-adrenergic receptor agonist and blocker.

F&#246;rster resonance energy transfer (FRET) microscopy is a powerful technique for real-time monitoring of signaling events in live cells using various biosensors as reporters. Here we describe how to build a customized epifluorescence FRET imaging system from commercially available components and how to use it for FRET experiments.

FRET microscopie pour Surveillance en temps réel des événements de signalisation dans les cellules vivantes en utilisant Biocapteurs unimoléculaires

F&ouml;rster resonance energy transfer (FRET) microscopy  continues to gain increasing interest as a technique for real-time monitoring  of biochemical ...

Monitoring Plasmid Replication in Live Mammalian Cells over Multiple Generations by Fluorescence Microscopy

Few naturally-occurring plasmids are maintained in mammalian cells. Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus (EBV) and Kaposi's Sarcoma-associated herpesvirus (KSHV), which cause multiple human malignancies 1-3. These two genomes are replicated in a licensed manner, each using a single viral protein and cellular replication machinery, and are passed to daughter cells during cell division despite their lacking traditional centromeres 4-8.
Much work has been done to characterize the replications of these plasmid genomes using methods such as Southern blotting and fluorescence in situ hybridization (FISH). These methods are limited, though. Quantitative PCR and Southern blots provide information about the average number of plasmids per cell in a population of cells. FISH is a single-cell assay that reveals both the average number and the distribution of plasmids per cell in the population of cells but is static, allowing no information about the parent or progeny of the examined cell.
Here, we describe a method for visualizing plasmids in live cells. This method is based on the binding of a fluorescently tagged lactose repressor protein to multiple sites in the plasmid of interest 9. The DNA of interest is engineered to include approximately 250 tandem repeats of the lactose operator (LacO) sequence. LacO is specifically bound by the lactose repressor protein (LacI), which can be fused to a fluorescent protein. The fusion protein can either be expressed from the engineered plasmid or introduced by a retroviral vector. In this way, the DNA molecules are fluorescently tagged and therefore become visible via fluorescence microscopy. The fusion protein is blocked from binding the plasmid DNA by culturing cells in the presence of IPTG until the plasmids are ready to be viewed.
This system allows the plasmids to be monitored in living cells through several generations, revealing properties of their synthesis and partitioning to daughter cells. Ideal cells are adherent, easily transfected, and have large nuclei. This technique has been used to determine that 84% of EBV-derived plasmids are synthesized each generation and 88% of the newly synthesized plasmids partition faithfully to daughter cells in HeLa cells. Pairs of these EBV plasmids were seen to be tethered to or associated with sister chromatids after their synthesis in S-phase until they were seen to separate as the sister chromatids separated in Anaphase10. The method is currently being used to study replication of KSHV genomes in HeLa cells and SLK cells. HeLa cells are immortalized human epithelial cells, and SLK cells are immortalized human endothelial cells. Though SLK cells were originally derived from a KSHV lesion, neither the HeLa nor SLK cell line naturally harbors KSHV genomes11. In addition to studying viral replication, this visualization technique can be used to investigate the effects of the addition, removal, or mutation of various DNA sequence elements on synthesis, localization, and partitioning of other recombinant plasmid DNAs.

A method of observing individual DNA molecules in live cells is described. The technique is based on the binding of a fluorescently tagged lac repressor protein to binding sites engineered into the DNA of interest. This method can be adapted to follow many recombinant DNAs in live cells over time.

Surveillance de la réplication plasmidique dans les cellules de mammifères en direct sur plusieurs générations par microscopie à fluorescence

Few naturally-occurring plasmids are maintained in mammalian cells.  Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus  (EBV) ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

Mesure des niveaux Heme synthèse dans des cellules de mammifères

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that provides an accurate and reproducible analysis. Probe-based chemistry offers the least background fluorescence as compared to other (dye-based) chemistries. Presently, there are several platforms available that use probe-based chemistry to quantitate transcript abundance. qPCR in a 96 well plate is the most routinely used method, however only a maximum of 96 samples or miRNAs can be tested in a single run. This is time-consuming and tedious if a large number of samples/miRNAs are to be analyzed. High-throughput probe-based platforms such as microfluidics (e.g. TaqMan Array Card) and nanofluidics arrays (e.g. OpenArray) offer ease to reproducibly and efficiently detect the abundance of multiple microRNAs in a large number of samples in a short time. Here, we demonstrate the experimental setup and protocol for miRNA quantitation from serum or plasma-EDTA samples, using probe-based chemistry and three different platforms (96 well plate, microfluidics and nanofluidics arrays) offering increasing levels of throughput.

Circulating microRNAs have recently emerged as promising and novel biomarkers for various cancers and other diseases. The goal of this article is to discuss three different probe-based real-time PCR platforms and methods that are available to quantify and determine the abundance of circulating microRNAs.

PCR en temps réel basée sur une sonde Approches de mesure quantitative des microARN

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that ...

In Situ Monitoring of Transiently Formed Molecular Chaperone Assemblies in Bacteria, Yeast, and Human Cells

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the JDP family could form transient heterocomplexes in eukaryotes to fine-tune substrate selection for the 70 kDa heat shock protein (Hsp70) chaperone-based protein disaggregases. The JDP complexes target acute/chronic stress induced aggregated proteins and presumably help assemble the disaggregases by recruiting multiple Hsp70s to the surface of protein aggregates. The extent of the protein quality control (PQC) network formed by these physically interacting JDPs remains largely uncharacterized in vivo. Here, we describe a microscopy-based in situ protein interaction assay named the proximity ligation assay (PLA), which is able to robustly capture&#160;these transiently formed chaperone complexes in distinct cellular compartments of eukaryotic cells. Our work expands the employment of PLA from human cells to yeast (Saccharomyces cerevisiae) and bacteria (Escherichia coli), thus rendering an important tool to monitor the dynamics of transiently formed protein assemblies in both prokaryotic and eukaryotic cells.

Cognate J-domain proteins cooperate with the Hsp70 chaperone to assist in a myriad of biological processes ranging from protein folding to degradation. Here, we describe an in situ proximity ligation assay, which allows the monitoring of these transiently formed chaperone machineries in bacterial, yeast and human cells.

Surveillance in Situ des assemblages de chaperon moléculaire s'est formé de façon transitoire dans les bactéries, les levures et les cellules humaines

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the ...