This work was supported by National Institutes of Health grants GM 100201 to I.F.S, and GM 048071 and GM 065830 to I.P.

Nous présentons des procédures détaillées pour l&#39;imagerie locales Ca 2+ événements dans les cellules SH-SY5Y neuroblastome humain. Ces procédures peuvent être adaptées à l&#39;image de Ca 2+ intracellulaire des signaux dans de nombreux types cellulaires 10/08. 1. Préparation des cellules <ol><li> Culture des cellules à imager selon les instructions figurant sur le site Internet de leur fournisseur. </li><li> Quelques jours avant l&#39;imagerie, les cellules de récolte cultivées dans un flacon de culture tissulaire en utilisant 1 ml de 0,25% de trypsine / EDTA (2-3 min). À la suite de détachement, ajouter un volume égal de milieu de culture pour inactiver la trypsine. </li><li> Effectuer une numération cellulaire en utilisant un hémocytomètre et de semences cellules dans des boîtes d&#39;imagerie à fond de verre à une densité de 3-7 x 10 4 cellules par boîte. Sélectionnez les cellules pour des expériences d&#39;imagerie quand ils atteignent 60% de confluence (2-3 jours). </li></ol> 2. Préparation des solutions et réactifs <ol><li> Préparer un Ca 2+ contenant tamponnée HEPESsolution de sel (Ca 2+ -HBSS), composée de (mM): NaCl 135, KCl 5,4, CaCl 2 2, MgCl 2 1, HEPES 10, 10 et glucose; pH = 7,4 à la température ambiante (TA) avec NaOH. Préparer Ca 2+ -HBSS sans l&#39;ajout de glucose et conserver à 4 ° C; ajouter à la solution de glucose immédiatement avant l&#39;utilisation. </li><li> Solubiliser les formes de la Ca 2+ indicateur fluorescent Cal-520 (1 mM) infiltrant les membranes, ci-IP 3 (200 uM), et EGTA (100 mM) dans du diméthylsulfoxyde (DMSO) contenant 20% de Pluronic F-127. Aliquoter ces réactifs dans des tubes Eppendorf et magasin, à l&#39;abri de l&#39;humidité et de la lumière, à -20 ° C. </li></ol> 3. Les cellules de chargement avec infiltrant les membranes Cal-520, ci-IP 3 et EGTA <ol><li> Préparer le &quot;tampon de chargement&quot; par dilution de solutions mères de infiltrant les membranes Cal-520 et CI-IP 3 à une concentration finale de 5 uM et 1 uM, respectivement, dans -HBSS Ca2 +. </li><li> Aspirer cumédias LTURE et rincer les cellules en remplaçant médias Ca 2+ -HBSS trois fois. </li><li> Retirer Ca 2+ -HBSS et incuber les cellules dans un tampon de chargement pendant 60 min à température ambiante dans l&#39;obscurité. </li><li> Éliminer le tampon de chargement et rincer les cellules en remplaçant médias Ca 2+ -HBSS trois fois. <ol><li> Si vous le souhaitez, à cette étape, les cellules de charge avec EGTA / AM en diluant la solution mère à une concentration finale de 5 uM dans Ca 2+ -HBSS et puis incuber les cellules pendant 30-60 min à température ambiante dans l&#39;obscurité. Après cette incubation, rincer les cellules trois fois avec Ca 2+ -HBSS avant de procéder à l&#39;étape 3.5. </li></ol></li><li> Incuber les cellules à Ca 2+ -HBSS de 30 min pour permettre la désestérification de réactifs chargés. </li><li> Immédiatement avant l&#39;imagerie, remplacer le Ca2 + avec -HBSS &quot;frais&quot; Ca 2+ -HBSS afin d&#39;éliminer tout colorant qui aurait fui dans le bain au cours de l&#39;incubation finale. </li></ol> 4. Ca 2+ </sup&gt; Acquisition d&#39;image <ol><li> Placer une petite goutte d&#39;huile d&#39;immersion sur l&#39;objectif 100X APO FRBR (NA 1,49). </li><li> Monter le plat d&#39;imagerie sur la scène du microscope inversé et amener les cellules au point en utilisant la lumière transmise. Il est important de fixer la capsule d&#39;imagerie pour empêcher tout mouvement pendant l&#39;enregistrement. </li><li> Illuminer les cellules avec un laser 488 nm afin d&#39;exciter Cal-520 et collecter la fluorescence émise (λ&gt; 510 nm) en utilisant une caméra EMCCD à grande vitesse. REMARQUE: Le logiciel qui fonctionne le microscope permet à l&#39;utilisateur de traduire une lentille de focalisation permettant le faisceau laser à introduire soit à l&#39;extrême bord de l&#39;ouverture de retour objectif (de la FRBR excitation) ou plus au centre (pour l&#39;excitation &quot;WF&quot;). </li><li> Utilisation du logiciel EMCCD, réduire le champ d&#39;imagerie des pleins 512 x 512 pixels afin de recueillir des données à la résolution temporelle nécessaire pour capturer locales Ca 2+ événements. REMARQUE: Par exemple, quad centreacquisition de 256 x 256 pixels accélère la vitesse d&#39;acquisition à 66 fps. Il est également possible d&#39;augmenter encore la fréquence d&#39;images de la caméra EMCCD 500 fps en utilisant une fonction du mode de récolte isolé. </li><li> Configurer le logiciel pour enregistrer automatiquement quelques secondes de l&#39;activité de base, offrir un flash UV aux cellules et continuer à enregistrer pour un autre 10 à 30 sec. REMARQUE: Un flash UV est fourni en utilisant une source de lumière à arc au xénon introduit à travers un miroir réfléchissant les UV dichroïque dans l&#39;orifice arrière du microscope. La durée et l&#39;intensité du flash UV est ajusté empiriquement pour évoquer une fréquence souhaitée des locaux Ca 2+ événements. </li><li> Enregistrer les fichiers que l&#39;image des piles pour l&#39;analyse hors ligne. </li></ol> 5. Analyse automatisée Ca 2+ Image NOTE: Il est possible de visualiser, traiter et analyser les données capturées en utilisant de nombreux logiciels commerciaux. Cependant, nous avons développé un algorithme pour automatiser l&#39;identification rapide d&#39;uneanalyse de Ca2 + d de signaux locaux. Une description détaillée des filtres spatiales et temporelles utilisées dans cet algorithme, la génération des AF / F 0 et l&#39;identification / analyse des routines peut être trouvée dans 11. Cet algorithme a été développé pour fonctionner sur la plate-forme open-source du logiciel Python, et peut être obtenu, avec l&#39;échantillon de données expérimentales et les instructions d&#39;utilisation détaillées, en envoyant un courriel de l&#39;auteur correspondant (ismith@uci.edu). <ol><li> Convertir des fichiers à importer dans l&#39;algorithme écrite sur mesure pour le format de fichier de la multi-plan. </li><li> Localisez le dossier contenant l&#39;algorithme d&#39;analyse et double cliquez sur le run.exe. </li><li> Sélectionnez le fichier .tiff à analyser. </li><li> Choisissez les paramètres d&#39;analyse dans la boîte de dialogue ouverte. Voir la figure 2A pour les paramètres par défaut pour les données d&#39;échantillon. </li><li> Déterminer le niveau de noir à être décalée par rapport à la pile de l&#39;image en déplaçant le curseur sur une partie du champ de vue qui ne nOT contient une cellule (voir la figure 2B) ou en saisissant manuellement un niveau de noir à l&#39;aide de l&#39;invite «Black Level Set &#39;. </li><li> Observez quatre fenêtres sur l&#39;écran après analyse par le logiciel (figure 2C-F). C est une image monochrome de repos fluorescence à partir de cellules en cours d&#39;analyse. Les carrés blancs superposés sur cette image sont des lieux d&#39;événements déterminés, que les positions barycentre de fonctions gaussiennes deux dimensions montés sur les événements. </li><li> Cliquez sur chaque lieu de l&#39;événement ou appuyez sur les touches de curseur pour faire défiler les événements. En faisant ainsi l&#39;arrière-plan soustrait, gaussien lissée changements de rapport de fluorescence (AF / F) 0 à chaque mise à jour de ces sites dans la fenêtre D. REMARQUE: Rouge souligné les événements sont déterminés à provenir de ce site particulier et non pas de «purge-through &#39;de l&#39;activité localisée à des sites adjacents. La trace bleue inférieure indique l&#39;endroit où le signal de fluorescence sur le site choisi dépassé la détectionseuil, alors que la ligne noire identifie les événements provenant de ce site particulier (correspondant à la couleur rouge des événements mis en évidence). </li><li> Cliquer sur un événement en surbrillance rouge pour mettre à jour la fenêtre E qui affiche l&#39;évolution temporelle de l&#39;événement, et la fenêtre F qui présente le profil spatial de l&#39;événement en moyenne sur son évolution dans le temps, ainsi que le profil spatial de la fonction gaussienne équipée. </li><li> Examiner manuellement les événements qui ont été identifiés à ce que les artefacts peuvent être rejetés de l&#39;analyse. Supprimer ces événements en cliquant droit sur le rouge souligné événements. NOTE: Dans nos mains, flux de Ca2 + par des voies simples IP 3 R évoque des signaux avec AF / F 0 environ 0,11, sont donc susceptibles d&#39;être de faux positifs des «événements» détectés sensiblement plus petites que cela. </li><li> Exporter des données en sélectionnant «Enregistrer sur Excel» ou exporter des données sur une base par cellulaire en dessinant autour de la cellule d&#39;intérêt et en sélectionnant «Enregistrer CellR17 ;. Note: Les données sont enregistrées dans le même dossier que la pile de l&#39;image originale. </li></ol>

<ol>
	<li>Berridge, M. J., Lipp, P., Bootman, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+versatility+and+universality+of+calcium+signalling.">The versatility and universality of calcium signalling.</a> Nat Rev Mol Cell Biol. 1 (1), 11-21 (2000).</li><li>Hill-Eubanks, D. C., Werner, M. E., Heppner, T. J., Nelson, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+signaling+in+smooth+muscle.">Calcium signaling in smooth muscle.</a> Cold Spring Harb Perspect Biol. 3 (9), a004549 (2011).</li><li>Hagenston, A. M., Bading, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+signaling+in+synapse-to-nucleus+communication.">Calcium signaling in synapse-to-nucleus communication.</a> Cold Spring Harb Perspect Biol. 3 (11), a004564 (2011).</li><li>Berridge, 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=Elementary+and+global+aspects+of+calcium+signalling.">Elementary and global aspects of calcium signalling.</a> J Physiol. 499 (Pt 2), 291-306 (1997).</li><li>Lipp, P., Niggli, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+hierarchical+concept+of+cellular+and+subcellular+calcium+signalling).">A hierarchical concept of cellular and subcellular calcium signalling).</a> Prog Biophys Mol Biol. 65 (3), 265-296 (1996).</li><li>Parker, I., Yao, Y., Ilyin, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+kinetics+of+calcium+liberation+induced+in+Xenopus+oocytes+by+photoreleased+inositol+trisphosphate.">Fast kinetics of calcium liberation induced in Xenopus oocytes by photoreleased inositol trisphosphate.</a> Biophys J. 70 (1), 222-237 (1996).</li><li>Minta, A., Kao, J. P., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+indicators+for+cytosolic+calcium+based+on+rhodamine+and+fluorescein+chromophores.">Fluorescent indicators for cytosolic calcium based on rhodamine and fluorescein chromophores.</a> J Biol Chem. 264 (14), 8171-8178 (1989).</li><li>Demuro, A., Parker, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+the+activity+and+localization+of+single+voltage-gated+Ca(2+)+channels+by+total+internal+reflection+fluorescence+microscopy.">Imaging the activity and localization of single voltage-gated Ca(2+) channels by total internal reflection fluorescence microscopy.</a> Biophys J. 86 (5), 3250-3259 (2004).</li><li>Smith, I. F., Parker, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+the+quantal+substructure+of+single+inositol+trisphosphate+receptor+channel+activity+during+calcium+puffs+in+intact+mammalian+cells.">Imaging the quantal substructure of single inositol trisphosphate receptor channel activity during calcium puffs in intact mammalian cells.</a> Proc Natl Acad Sci U S A. 106 (15), 6404-6409 (2009).</li><li>Reissner, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+postsynaptic+mechanism+for+heterosynaptic+sharing+of+short-term+plasticity.">A novel postsynaptic mechanism for heterosynaptic sharing of short-term plasticity.</a> J Neurosci. 30 (26), 8797-8806 (2010).</li><li>Ellefson, K. S., Parker, B., Smith, I., 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=An+algorithm+for+automated+detection,+localization+and+measurement+of+local+calcium+signals+from+camera-based+imaging.">An algorithm for automated detection, localization and measurement of local calcium signals from camera-based imaging.</a> Cell Calcium. , (2014).</li><li>Smith, I. F., Wiltgen, S. M., Parker, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+puff+sites+adjacent+to+the+plasma+membrane:+Functional+and+spatial+characterization+of+calcium+signaling+in+SH-SY5Y+cells+utilizing+membrane-permeant+caged+inositol+trisphosphate.">Localization of puff sites adjacent to the plasma membrane: Functional and spatial characterization of calcium signaling in SH-SY5Y cells utilizing membrane-permeant caged inositol trisphosphate.</a> Cell Calcium. 45, 65-76 (2009).</li><li>Shuai, J., Parker, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+single-channel+recording+by+imaging+calcium+flux+through+individual+ion+channels:+theoretical+considerations+and+limits+to+resolution.">Optical single-channel recording by imaging calcium flux through individual ion channels: theoretical considerations and limits to resolution.</a> Cell Calcium. 37 (4), 283-299 (2005).</li></ol>

The authors declare that they have no competing financial interests.

Nous décrivons ici les protocoles pour l&#39;imagerie locales Ca 2+ événements dans des cellules de mammifères cultivées en utilisant fluorescentes Ca 2+ indicateurs. En outre, nous décrivons un algorithme avec une interface utilisateur intuitive qui automatise l&#39;identification et l&#39;analyse des données acquises. La procédure décrite ici utilisent le Ca 2+ fluorescente indicateur Cal-520, mais beaucoup d&#39;autres colorants Ca 2+ sensibles comme Fluo-3, Fluo-4...

Les ions calcium (Ca2 +) régulent ubiquitaire un large éventail de processus biologiques, y compris l&#39;expression des gènes, la sécrétion et des changements durables dans la plasticité synaptique 1. Un moyen par lequel Ca 2+ peut agir de manière diversifiée est à travers les différentes configurations spatiales et temporelles de Ca 2+ signale une cellule peut générer. Par exemple, les élévations mondiaux dans cytosolique [Ca 2+] déclenchement de la contraction dans le tissu musculaire lisse 2 alors plus petit, élévations transitoires localisées (locales Ca2 +) microdomaines stimuler l&#39;expression de gène essentiel pour l&#39;apprentissage et la mémoire 3. Cytosolique libre [Ca 2+] est maintenue à ~ 100 nM au repos, mais peut rapidement se élever à plusieurs micro-molaire suivante l&#39;afflux de Ca2 + dans le cytosol par Ca 2+ canaux ioniques -permeable situés dans la membrane plasmique et par la libération de Ca 2+ intracellulaire de stores. Notre laboratoire se concentre sur le récepteur de l&#39;inositol 1,4,5-triphosphate (IP 3 R), qui forme un canal de libération 2+ Ca situé dans la membrane du réticulum endoplasmique (RE). Lors de la liaison à la fois de IP3 et Ca2 + cytosolique à l&#39;activation des sites du récepteur, le canal R IP 3 se ouvre pour libérer Ca2 + séquestré dans la lumière du RE. La libération de Ca 2+ peut rester dans l&#39;espace restreint à un petit groupe de IP 3 Rs pour générer un microdomaine cytosolique locale de Ca2 + (Ca 2+ feuilletée 4) ou, en fonction de la proximité des pôles voisins de IP 3 Rs, peut propager dans une cellule en recrutant plusieurs sites feuilletées par un processus de Ca 2+ induite par Ca 2+ -release (CICR) 5,6. L&#39;introduction d&#39;indicateurs les colorants de petite molécule fluorescente Ca développé par Roger Tsien 7, couplé avec la microscopie avancée Imaging techniques, a grandement facilité notre compréhension de Ca 2+ signalisation. Les progrès récents en caméras utilisées pour la microscopie permettent désormais pour l&#39;imagerie transitoires locales Ca 2+ événements tels que bouffées avec une résolution spatiale et temporelle sans précédent. Actuellement disponibles caméras EMCCD permettent imagerie avec 128 x 128 pixels à 500 images&gt; -1 sec (fps) et la nouvelle génération de semi-conducteur complémentaire à oxyde de métal (CMOS) caméras offrent une résolution de pixels, et la vitesse encore plus rapide au détriment de légèrement plus élevé des niveaux de bruit. En collaboration avec réflexion totale interne (FRBR) microscopie, il est maintenant possible d&#39;une seule image Ca 2+ Les événements de canaux 8,9. Cette approche permet l&#39;imagerie de centaines de canaux / événements simultanément, tout en générant de grands ensembles de données (environ 1 Go par min) qui rendent le traitement manuel, l&#39;identification visuelle et l&#39;analyse impraticable et placer un fardeau sur le développement d&#39;algorithmes automatisés. 2+ signaux dans les cellules de mammifères en utilisant intactes fluorescentes Ca 2+ indicateurs. Nous démontrons en outre un algorithme développé dans l&#39;environnement Python open-source qui automatise l&#39;identification et l&#39;analyse de Ca 2+ événements locaux imagés à la fois par la FRBR et large champ épi-fluorescence conventionnelle (WF) microscopie. Bien que nous décrivons ces approches dans le contexte de la PI 3 -generated signaux Ca2 +, ils sont facilement prête pour étudier les changements locaux dans cytosolique [Ca 2+] issues d&#39;une variété de Ca 2+ canaux ioniques -permeable situés soit dans la surface ou membrane d&#39;organites intracellulaires 10/08.

<table border="0" cellpadding="0" cellspacing="0" width="1055">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:813px;">Name</td>
			<td style="width:124px;">Company</td>
			<td style="width:119px;">Catalogue No.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Human Neuroblastoma SH-SY5Y cells</td>
			<td>ATCC</td>
			<td>CRL-2266</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cal-520/AM</td>
			<td>AAT Bioquest Inc.</td>
			<td>21130</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">ci-IP3 (D-23-O-Isopropylidene-6-O-(2-nitro-4.5-dimethoxy)benzyl-myo-Inositol 145-trisphosphate-Hexakis(propionoxymethyl)</td>
			<td>SiChem</td>
			<td>cag-iso-2-145-10</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMSO/20% pluronic F127</td>
			<td>Invitrogen</td>
			<td>P-3000MP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EGTA/AM</td>
			<td>Invitrogen</td>
			<td>E-1219</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">35 mm glass-bottom imaging dishes</td>
			<td>MatTek</td>
			<td>P35-1.5-14-C</td>
		</tr>
	</tbody>
</table>

imaging local ca2+ signals in cultured mammalian cells

Cytosolique ions Ca 2+ réglementer de nombreux aspects de l&#39;activité cellulaire dans presque tous les types de cellules, le contrôle des processus aussi variés que la transcription des gènes, excitabilité électrique et la prolifération cellulaire. La diversité et la spécificité de Ca 2+ signalisation dérive de mécanismes par lesquels Ca 2+ signaux sont générés pour agir sur des échelles de temps et d&#39;espace différentes, allant des oscillations et des vagues large cellulaires survenant au cours des périodes de minutes à transitoires Ca2 + microdomaines locales (Ca 2+) bouffées millisecondes durables. Les progrès récents dans électrons multipliés CCD (EMCCD) caméras permettent désormais pour l&#39;imagerie des signaux Ca 2+ locaux avec une résolution spatiale de 128 x 128 pixels à des taux de&gt; 500 cadres sec -1 (fps). Cette approche est très parallèle et permet la surveillance simultanée de centaines de chaînes ou d&#39;autres sites feuilletées dans une seule expérience. (1 pe Cependant, les grandes quantités de données générées ca. Gbr min) de rendre l&#39;identification visuelle et l&#39;analyse de Ca 2+ locale événements impraticable. Ici, nous décrire et démontrer les procédures pour l&#39;acquisition, la détection et l&#39;analyse des IP locale 3 médiée Ca 2+ signaux dans les cellules mammifères intactes chargés de Ca 2+ indicateurs utilisant à la fois grand champ épi-fluorescence (WF) et la réflexion interne totale fluorescence (FRBR) microscopie. En outre, nous décrivons un algorithme développé au sein de l&#39;open-source environnement logiciel Python qui automatise l&#39;identification et l&#39;analyse de ces signaux locaux Ca 2+. L&#39;algorithme localise les sites de Ca 2+ de presse avec une résolution sous-pixel; permet d&#39;avis utilisateur des données; et délivre séquences temporelles de signaux de rapport de fluorescence avec amplitude et données cinétiques dans un tableau Excel compatible.

Figure 1A montre une image WF de repos Cal-520 fluorescence dans les cellules de neuroblastome SH-SY5Y humains également chargé avec 3 ci-IP. L&#39;exposition de ces cellules à un flash 100 UV msec à la photo-presse i-IP 3 a suscité transitoires Ca2 + bouffées sur des sites discrets (relevées par les cercles blancs sur la figure 1A). traces de fluorescence mesurée à ces sites ont montré une phase de montée rapide en raison d&#39;ouvertures de ...

Watch this Scientific Journal Video about Imaging Local Ca2+ Signals in Cultured Mammalian Cells at JoVE.com

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

Imagerie Ca locale<sup&gt; 2+</sup&gt; Signaux de cellules de mammifères cultivées

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

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Imaging Local Ca2+ Signals in Cultured Mammalian Cells

10.7K vues.  University of California, Irvine. Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

Vidéo: Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

Actually what I'm gonna do, mount it in here first and then swap out You. So now I'm gonna aspirate try and be as gentle as possible.Okay. A few seconds just holding.Okay.Alright.So the important features of our lifestyle imaging grid are that, so it's an inverted scope, which means the objective, the samples here in the objective is underneath. So the objective is pointing up and the whole scope, or practically the whole, practically the whole scope, including the objectives and the sample are at 37 degrees. So everything in this plastic box is at 37 degrees.And right on top of the stage we have an air and CO2 mix. That is 5%CO2. So the idea is that the sample is actually in a cell, it's like a cell incubator.And so we have, this is our mixer for the air and the CO2 and, and it gets humidified and heated and then piped onto the, the sample on the stage.Okay. And today we're just gonna be doing fluorescence one channel GFP fluorescence. This is the hose that the CO2 and 5%CO2 goes in and it just fits into the stage holder.That's important. And then we can close these, take these down, and then we go to our first position. And what I'm gonna do is go to the first field, which is my control sample, and I'll pick positions based on what I see on the screen.So I just make sure I actually look into the microscope in each new, well, because the focal plane for each of the wells is different, the, the dish is not perfectly flat on the bottom. So you will need to make fine adjustments when you move from well to the well. So right now I'm on the first, well, what I'll do is I'll focus, so I just hit, I just told, I just turned on the light, the transmitted light for the microscope.And I have make sure all the light is going to the eye pieces. And so I just look in, make sure I get a nice clean field. And then what I'll do is turn that light off, turn the fluorescence light on, make sure the cells that I'm looking at look healthy and they're not apoptotic.And, and I turn that off. And what I do next is send, so now I'm gonna start picking positions, which means I have to te send the light path to the camera. This is the camera right here.So I tell the microscope to send all the camera, they don't see anything in the eye pieces. What I'll do is in the software controlling the microscope, I'll choose the right filter set, which for our purposes is psy. And then set an exposure time.And I know these cells very well and I know that they take a 0.2 second exposure time. So I tell, that's what I tell the software to do. And what I'll do is hit focus.This is, and it's basically just alive. The, A light will turn on and you can, oh, I don't know if you can see cells, but the reason this is a good field is because there's, all the cells are, even in terms of their fluorescence, There are no apoptotic cells. Apoptotic cells tend to be very bright and there's no dirt or schmutz in the field.And you can see that they're actually, this is a metaphase cell and that's a metaphase cell, that's a cell that's probably just exited mitosis. And I can just tell that because it looks like those are, those two sets of chromatin are very nicely oriented to each other. Like they just exited an face.So that's a good field. So, and now that I know that the exposure time and the filter set is correct, and this is gonna be software specific, I'll go and I'll take positions and now the software has memorized the X, Y, Z position of that field. And then when I do pick another field, it's, and I'll, so when I'm picking, when I'm picking new fields, but in the same, well, I'll use the, I'll move the i'll, I'll turn the shutter, I'll open the shutter and I'll look to see what I see on the screen and I'll pick fields based on that versus looking in the field.So I don't see any of my tonics in there. But again, even nicely centered, these cells will go through my PS I continue, here's a good example. Here's a good example of what I would avoid that, see how bright that is?That's very bright. That's probably remnants of an apoptotic cell. And the reason I would avoid that is because since it's so bright, when the software auto focuses, it will autofocus just on that and it won't be in the same plane as all these other cells.And so I won't get any information from that field. That looks nice. There's a, this is a pro metaphase right here, That's A metaphase.So, and I'm gonna take three positions per well. So that was three positions and now I'm going to go to my next Well, so you can, so what I will probably do is take images every 10 minutes. So it's a compromise.Lifestyle imaging is always a compromise, right? You have to image, you have to keep the cells healthy, especially if you're interested in mitosis. So you have to minimize their exposure to the fluorescent line.And, but you wanna get information. So you have to take images, you have to, so you have to compromise, you have to make a choice about what you want to capture, what kind of information you wanna capture. For our purposes, mitosis tends to take about an hour.So if we're taking 10 minute intervals, that's pretty good. We see prophase, we see metaphase, we see anaphase, and we can, we can see when there are, there are treatments or drugs or siRNAs that affect those specific cell cycle stages. So for our purposes, that's enough, but we always want more.We always want more positions or finer time and interval or something like that. So it's a compromise. So, and then I'll hit start.And so our software, it does an autofocusing pass on the first pass. So once I hit start, it should start auto autofocusing and you'll see the shutter open, open and close between auto focusing. So I'll hit start.So that's the shutter opening and closing. The Z motor is engaging and it's actually trying to find the plane with the most contrast, which will be the most in focus. You can actually see on the screen going in and out of finding the best plane of focus that might be worrisome.So what it does is it, it drives the Z motor to find the best, the most in focus plane focus, well the most in image. And then it acquires picture there and then it learns that Z position. And then we'll use that Z position for the next 12 passes.And then we'll do the auto focusing pass again. What Kind of experiments do they do? Okay, so our lab is mostly interested in understanding what regulates mitic progression, or at least I'm most interested in understanding that.So with live cell imaging and especially our multi position, long-term imaging scope, you can take, you can film cells for two or three days entering anding mitosis, and you can image multiple rounds of mitosis so you can understand. And then you can put drugs or RNAs or any kind of perturbations on the cells that you would like to study. So you can understand how, how those drugs or, or genes are, are important in regulating mitotic progression.Because it's not fixed cell work and live cell work, you can understand what, when you need to have a perturbation to affect mitosis or what stage of mitosis is affected. So it's really powerful in that respect. What, what are the main technical elements of this experiments?What are the possible problems? Well, the biggest problem besides getting the rig set up, getting the rig set up is not trivial. A lot of people have the rig, but just making sure it works well for your application.The financial element is not, you know, is not small. So there's the setting up of the apparatus and then, and then once you gather the data, there's a real data analysis issue. It takes these movie, if, so, if you're gathering 40 movies each with 30 cells, so you have 40 in 40 fields that you just filmed and each of those fields has 30 cells, then you have to sit down with each one of those movies and look at each one of those cells in that movie.So it takes a really long time to do data analysis. And that's, so it takes a week to analyze data that it took you two days to collect. So there's a, that's a very big obstacle in terms of gathering lots of data.So it's not, while it's high throughput data collection, it's not high throughput analysis. So what would you make to make this experiment really high throughput? So our lab is in the process of developing a program that does the data analysis automatically.And it's a pretty decent program. It's not perfect. The human eye is always better, but it's getting there.So What are the interesting application of this experiment? Maybe other fields you can mention? Besides cell division?You mean for the, oh, for the live cell imaging? Yes.Well, you can do cell motility assays. You can, depending on the, the microscopy that's involved, you can, you know, look at how cells, you could look at tumor development if you really wanted to.But there, you, you're, if as long as you have the right imaging set up, you can, you're pretty much not limited in terms of anything. Our scope happens to be just a wide field and we use 20 by 20 x objective. So it's a pretty low resolution set up, but that's sufficient for what we need to see.So there are, but there definitely aren't, you wouldn't be limited. Anything that has any dynamic kind of experiment, which is biology, it would be, would really benefit from lifestyle imaging. So calcium imaging, although you couldn't do it on our apparatus, that's a live cell.You definitely need live cell imaging for that. Like I said, patient assays, motility assays, those kinds of things would really benefit from live cell imaging. Would you like to tell a few words about your previous experience?How long do you work on these techniques? What do you you work on before? Wow.So in my PhD I didn't work.I did a lot of confocal microscopy, but no lifestyle imaging. And so during my postdoc I was, I helped Dr.King get this apparatus that, that we just used to set up these fragment, get that up and running. And, and so during my postdoc, I had a lot of experience with both widefield with TimeLapse imaging on our scope, which is the widefield and also TimeLapse imaging with confocal microscopy.And I would say that it's really powerful. You get a lot of information, but it's not easy to get working perfectly. So it really requires a lot of attention and a lot of attention to details at every little step of the process.And, and then you still have problems. So it's a tough application, but you can really get, if, if it's what you need to have to, to study your problem, your scientific problem, then it, it's invaluable in that respect, but it's tough. So find someone who knows what they're doing and use them.That's my advice. So good.

Cellules d&#39;imagerie à long terme des mammifères en utilisant la microscopie à champ large

Recherche

Biologie

Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  Vital fluorescent probes, particularly green fluorescent protein (GFP) have revolutionized cell biology stemming from the ability to label specific intracellular compartments and cellular structures. For example, the live imaging of GFP (and its spectral variants) chimeras have allowed for a dynamic 
analysis of the cytoskeleton, organelle transport, and membrane dynamics in a multitude of organisms and cell types [1-3].  Although live imaging has become prevalent, this approach still poses many technical challenges, particularly in primary cultured neurons.  One challenge is the expression of GFP-tagged proteins in post-mitotic neurons; the other is the ability to capture fluorescent images while minimizing phototoxicity, photobleaching, 
and maintaining general cell health.  Here we provide a protocol that describes a lipid-based transfection method that yields a relatively low transfection rate (~0.5%), however is ideal for the imaging of fully polarized neurons.  A low transfection rate is essential so that single axons and dendrites can be characterized as to their orientation to the cell body to confirm directionality of transport, i.e., anterograde v. retrograde.  Our approach to imaging 
GFP expressing neurons relies on a standard wide-field fluorescent microscope outfitted with a CCD camera, image capture software, and a heated imaging chamber. We have imaged a wide variety of organelles or structures, for example, dense-core vesicles, mitochondria, growth cones, and actin without any special optics or excitation requirements other than a fluorescent light source. Additionally, spectrally-distinct, fluorescently 
labeled proteins, e.g., GFP and dsRed-tagged proteins, can be visualized near simultaneously to characterize co-transport or other coordinated cellular events.  The imaging approach described here is flexible for a variety of imaging applications and can be adopted by a laboratory for relatively little cost provided a microscope is available.

Live cell imaging is of particular utility when studying the dynamics of organelle trafficking. Here we describe a protocol for live imaging of dense-core vesicles in cultured neurons using wide-field fluorescence microscopy. This protocol is flexible and can be adapted to image other organelles such as mitochondria, endosomes, and peroxisomes.

Imagerie en temps réel du noyau dense de vésicules cultures primaires neurones de l&#39;hippocampe

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  ...

Techniques for Imaging Ca2+ Signaling in Human Sperm

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca2+-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca2+-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca2+ response as % change in fluorescence is obtained.

Techniques for Imaging Ca2+ Signaling in Human Sperm

Stimulus-evoked [Ca2+]i signals of individual human sperm are assessed. Motile cells are loaded with Ca2+-sensitive fluorescent dye (AM-ester method) and immobilised in a perfusable chamber. Cells are imaged by time-lapse fluorescence microscopy and stimulated via the perfusing medium. Responses of single cells (or regions) are analysed offline using Excel.

Techniques d&#39;imagerie Ca 2 + Signalisation dans le sperme humain

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in ...

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 ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

Basées sur la fluorescence de mesure de l&#39;entrée du calcium magasin fonctionnant dans des cellules vivantes: à partir de cellules cancéreuses en culture de fibre musculaire squelettique

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

Analyse de la situation du cycle cellulaire dans les cellules de mammifères

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ imaging data is time consuming and subject to bias. Automated signal analysis algorithms based on region of interest (ROI) detection have been implemented for one-dimensional line scan measurements, but there is no current algorithm which integrates optimized identification and analysis of ROIs in two-dimensional image sequences. Here an algorithm for rapid acquisition and analysis of ROIs in image sequences is described. It utilizes ellipses fit to noise filtered signals in order to determine optimal ROI placement, and computes Ca2+ signal parameters of amplitude, duration and spatial spread. This algorithm was implemented as a freely available plugin for ImageJ (NIH) software. Together with analysis scripts written for the open source statistical processing software R, this approach provides a high-capacity pipeline for performing quick statistical analysis of experimental output. The authors suggest that use of this analysis protocol will lead to a more complete and unbiased characterization of physiologic Ca2+ signaling.

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Here a novel region of interest analysis protocol based on sorting best-fit ellipses assigned to regions of positive signal within two-dimensional time lapse image sequences is demonstrated. This algorithm may enable investigators to comprehensively analyze physiological Ca2+ signals with minimal user input and bias.

Analyse automatisée de dynamique Ca<sup&gt; 2 +</sup&gt; Signaux de séquences d&#39;images

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

siRNA contrôle pour la ubiquitine et les régulateurs du système ubiquitine-comme des voies biologiques dans des cellules de mammifères en culture

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

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 ...

Massively Parallel Reporter Assays in Cultured Mammalian Cells

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. The potential throughput of this assay has, however, been limited by the need to individually clone and assay the activity of each sequence on interest using protein fluorescence or enzymatic activity as a proxy for regulatory activity. Advances in high-throughput DNA synthesis and sequencing technologies have recently made it possible to overcome these limitations by multiplexing the construction and interrogation of large libraries of reporter constructs. This protocol describes implementation of a Massively Parallel Reporter Assay (MPRA) that allows direct comparison of hundreds of thousands of putative regulatory sequences in a single cell culture dish.

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. Coupling candidate regulatory elements to reporter genes that carry identifying sequence tags enables massive parallelization of these assays.