Le financement de ce projet a été fournie par la National Science Foundation accorde OCE1029607 (XM) et MCB0702125 (au MAM) et la Gordon and Betty Moore Foundation (au MAM).

1. Traitement des échantillons d&#39;eau <ol><li> Filtrer l&#39;eau 10L de l&#39;environnement grâce à une membrane filtrante à pores um de taille pour enlever les grosses particules et bacteriovores. Recueillir le filtrat de l&#39;eau dans une bonbonne. </li><li> Transfert de 36 ml de chaque filtrat dans 3 tubes Eppendorf stériles (50 ml) contenant 4 ml solution fraîchement préparée de paraformaldéhyde (PFA; 10%). Incuber pendant 2 heures à température ambiante pour préserver les cellules. Recueillir cellules sur 0,22 um de-la taille des pores des filtres à membrane blanche par filtration sous vide. Lavez le filtre en faisant passer 10 ml de tampon phosphate salin (PBS) à travers le filtre par filtration sous vide. Étiqueter les filtres de contrôles négatifs et de les stocker à -20 ° C. </li></ol> Étapes 1.3 à 1.4 sont facultatives pour établir DOC-limitée conditions. <ol start="3"><li> Ajouter un mélange d&#39;azote et de phosphore inorganiques (5 uM NH 4 Cl, 5 uM NaNO 3, et 1 uM NaH 2 PO 4 de concentration finale) dans la tourie. </li><li> Incuber dans l&#39;obscurité à température in situ pendant 48 heures avec agitation occasionnelle. </li></ol> 2. Microcosme création et l&#39;incubation <ol><li> Remplissez chacune des six flacons de verre de 1 L avec 800 ml d&#39;eau de l&#39;échantillon de la bonbonne de l&#39;étape 1.4 pour établir des microcosmes. Ajouter BrdU (10 uM, concentration finale) de chaque microcosme. Mélangez bien. </li><li> Ajouter 1 ml de modèle composé solution DOC dans trois des microcosmes, ces servira modifications DOC. Ajouter 1 ml de PBS stérile dans les trois restantes microcosmes, ces servira sans plus de contrôle. </li><li> Incuber tous les microcosmes dans un agitateur incubateur et incuber dans l&#39;obscurité à température in situ tout en agitant à 100 rpm. </li><li> Recueillir 36 ml d&#39;échantillon d&#39;eau de chaque microcosme et transférer dans des tubes de 50 ml Eppendorf stériles à des points de temps de 0, 8, 16 et 24 heures. Immédiatement ajouter 4 ml de frais PFA (10%) pour des tubes de prélèvement et incuber pendant 2 heures à température ambiante pour préserver les cellules. </li><li> Filtre préservé cellules par 0,22 um-la taille des pores des filtres à membrane en polycarbonate. Laver les filtres avec 10 ml de PBS. Procéder immédiatement à l&#39;étape suivante ou stocker les filtres à -20 ° C. </li></ol> 3. En immunodétection in situ pour l&#39;incorporation de BrdU <ol><li> Décongeler les filtres (échantillons DOC modifiée, sans plus de contrôle et les contrôles négatifs) à température ambiante. </li><li> Appliquer 1 ml de solution de lysozyme [10 mg / ml de lysozyme de blanc d&#39;œuf dans 100 mM Tris, 50 nM EDTA (pH = 8)] 4 pour couvrir les cellules bactériennes sur le filtre. Incuber à température ambiante pendant 30 minutes. Lavez le filtre en faisant passer 10 ml de PBS à travers elle sous aspiration. </li><li> Ajouter 1 ml de solution de protéinase K [2 mg / ml de protéinase K dans 100 mM Tris, 50 nM EDTA (pH = 8)] 4 pour couvrir les cellules bactériennes sur le filtre. Incuber à température ambiante pendant 30 minutes. Lavez le filtre en faisant passer 10 ml de PBS à travers elle sous aspiration. </li><li> Ajouter une solution de exonucléase ml [exonucléase III (50 U / ml) dans 5 mM de MgCl2 et 50 mM Tris-HCl] 5 pour couvrir les cellules bactériennes sur le filtre. Incuber à 37 ° C pendant 30 minutes. Lavez le filtre en faisant passer 10 ml de PBS à travers elle sous aspiration. </li><li> Assemblez chambres d&#39;incubation cadre joint (Bio-Rad) selon les instructions du fabricant. </li><li> Trancher le filtre en huitièmes à l&#39;aide d&#39;une lame stérile sur une consommation d&#39;alcool nettoyé la surface sèche. </li><li> En utilisant des pinces, placer une huitième section d&#39;un filtre dans une chambre d&#39;incubation assemblés cadre joint (huit cadres joint chambres sont nécessaires pour chaque échantillon de filtre). Le côté de la section de filtre qui contient des cellules doit faire face à la hausse. L&#39;humidité à l&#39;arrière du filtre permet le bâton de filtre à la diapositive. Si le filtre devient sèche, appliquer une petite goutte (2 pi) du dih 2 O dans le centre de la chambre avant de placer la section du filtre sur la diapositive. </li></ol> Étapes 03.08 à 03.20 utilisent les réactifs de la ROCHE En Kit prolifération cellulaire in situ, fluos en suivant les procédures de modification des instructions du fabricant. Sauf pour PBS, tous les réactifs sont fournis dans le kit. <ol start="8"><li> Appliquer suffisamment de tampon d&#39;incubation (0,5% d&#39;albumine sérique bovine, 0,1% de Tween 20 dans du PBS), pour bien couvrir toute la section de filtre dans la chambre d&#39;incubation sans causer de débordement fois le sceau est appliqué. </li><li> Placez un couvercle en polyester cadre sur le cadre de la chambre d&#39;incubation. Appuyez pour bien sceller la chambre d&#39;incubation. Eviter les bulles d&#39;air au-dessus du filtre. Incuber les chambres scellées pendant 10 min à température ambiante dans l&#39;obscurité. </li><li> Retirer le joint de polyester et d&#39;ouvrir des chambres d&#39;incubation. (Remarque: l&#39;ouverture des chambres, parfois se tirer vers le haut le sceau cadre de la diapositive Dans ce cas, de préparer une nouvelle chambre pour les étapes suivantes..) </li><li> Pipeter le tampon d&#39;incubation d&#39;un coin de la chambre. Eviter le contact avec le filtre. </li><li> Lavez le filtre en douceur de pipetage 100 ul de PBS dans ethors de la chambre 3 fois. </li><li> Préparer anti-BrdU-fluos solution de travail en suivant les étapes recommandées par le fabricant immédiatement avant utilisation. </li><li> Pipeter 120 pi anti-BrdU-fluos solution de travail sur le filtre, en prenant soin de façon uniforme couvrir toute la surface. </li><li> Refermer la chambre avec une housse de polyester neuf. Eviter les bulles d&#39;air sur le dessus du filtre. Incuber la chambre dans l&#39;obscurité à 37 ° C pendant 3 heures. Cette étape sera l&#39;étiquette BrdU incorporé l&#39;ADN in situ avec de l&#39;isothiocyanate de fluorescéine (FITC). </li><li> Retirez le couvercle en polyester et d&#39;ouvrir la chambre d&#39;incubation. Pipeter la solution anti-BrdU-fluos de travail. Lavez le filtre 3 fois avec du PBS. </li><li> Transférer le filtre de la chambre d&#39;incubation d&#39;une surface stérile. Trancher la section filtre en petits morceaux à l&#39;aide d&#39;une lame stérile. </li><li> Transférer les morceaux de filtre en 2 microtubes ml, contenant chacun 1 ml de PBS. Étroitement hermétiquement le tube et le joint avec du parafilm. Incuber à 37 ° C et 200 rpm pendant 10 min. </li><li> Fixez les tubes sur un Vortexer et le vortex à la vitesse maximale pendant 5 minutes. Déposer le surnageant dans un tube stérile de 15 ml Eppendorf. Répétez les étapes d&#39;incubation et de vortex pour 5 fois plus. Combinez le surnageant dans le même tube de 15 ml Eppendorf pour chaque échantillon. Typiquement, 80% de cellules peuvent être récupérés dans la suspension. </li><li> Stocker le surnageant de cellules en suspension à 4 ° C. Trier dans les 2 jours. </li></ol> 4. Analyse FACS Une procédure pour un cytomètre de flux BD FACSAria et du logiciel correspondant est décrit ici. <ol><li> Optimiser le réglage du débit étapes suivantes cytomètre recommandé par le fabricant. Cela implique: le réglage du contrôle d&#39;amplification de définir les paramètres requis et breakoff le sweet spot, optimisation du délai laser et zone de facteurs d&#39;échelle pour la pression de la gaine expérimenter et optimiser les réglages pour le FSC et SSC tensions, seuil FSC, FSC de fluorescence d&#39;échelle, la fluorescence tensions PMT , etc </li><li> Exécuter des échantillons de contrôle négatif sur le cytomètre en flux (FCM) en fonction de l&#39;intensité de fluorescence du FITC et dispersent de côté (SSC). Augmenter le seuil FITC jusqu&#39;au pas de cellules dans les contrôles négatifs peuvent être visualisés par l&#39;affichage d&#39;acquisition FITC-SSC. </li><li> Exécuter des échantillons de contrôle sans plus et d&#39;examiner le mode de distribution de 10 000 cellules en fonction de FITC et l&#39;acquisition de la SSC. Définir une porte pour enfermer toutes les cellules et de les désigner comme des «cellules de basse intensité» (LIS). </li><li> Exécuter DOC-amendé échantillons et d&#39;examiner le modèle de distribution de 10.000 cellules basées sur FITC et SSC. Certaines cellules apparaissent dans la grille prédéfinie LI. Définir une autre porte pour enfermer les cellules qui ont une plus grande intensité de fluorescence (HIS) que Lis. Acquérir les statistiques pour voir l&#39;abondance relative des cellules fermée. </li><li> Trier les cellules dans des tubes HI collection contenant 500 ul de PBS à «purifier 1 goutte&quot; mode. Terminez le tri lorsque le nombre de ses atteint 500 000 chefs d&#39;accusation. </li></ol> 5. Filtre amplification par PCR des gènes ARNr 16S Filtre les procédures de PCR sont modifiées de Kirchman et al. 6 <ol><li> Filtre cellules triées sur un blanc, 0,22 um-pores de taille, 25 mm de diamètre, membrane en polycarbonate filtre. Coupez le bord du filtre qui ne contient pas de cellules bactériennes en utilisant une lame stérile. Trancher le filtre en 8 morceaux égaux de taille. </li><li> Placez un seul morceau de filtre dans un tube de réaction PCR, avec les cellules vers l&#39;intérieur du tube. Ajouter de l&#39;eau 45 ul de grade PCR dans le tube de réaction PCR. Plonger le filtre entièrement dans l&#39;eau. </li><li> Ajouter 2 ROCHE illustra PuReTaq Read-To-Go Perles PCR dans le tube de réaction PCR, brièvement au vortex. Ajouter 2 ul de chacune des avant et arrière du gène ARNr 16S amorces (0,4 uM concentration finale pour chaque amorce), tels que 27F et 1492R 7, dans chacun des tubes de réaction PCR. </li><li> (Facultatif) Ajoutez 1 microlitre bovins solution de sérum albumine (BSA, la concentration finale est 30μg/100 ul) au mélange réactionnel de PCR pour aider à l&#39;amplification des inhibiteurs de s&#39;adsorber. Si l&#39;on choisit de ne pas ajouter de BSA, ajouter 1 microlitre d&#39;eau à la place. </li><li> Effectuer l&#39;amplification par PCR sur un cycleur thermique. Une touche bas programme de PCR est recommandée, qui a la température de recuit séquentielle diminuant de 62 à 52 ° C par 1 ° C par cycle de 11 cycles, suivie par 15 cycles avec une température de recuit de 52 ° C. Tous les cycles comprennent dénaturant (à 95 ° C), le recuit (à 62 à 52 ° C), et l&#39;extension (à 72 ° C) les étapes d&#39;une durée de 50 ans. Un initiale de 3 min de dénaturation et dernière étape d&#39;extension de 10 min est également inclus dans le programme de PCR. </li><li> Confirmer l&#39;amplification par PCR par électrophorèse sur un bromure d&#39;éthidium teinté gel à 1% d&#39;agarose. Accise amplicons PCR à partir du gel et de nettoyer avec le kit QIAGEN QIAquick gel extraction. </li><li> Effectuer deux autres amplifications PCR pour chaque échantillon, chaque fois que l&#39;utilisation d&#39;une nouvelle section de filtre. Après un gel de PCR Purificatile, la piscine des amplicons du même échantillon ensemble. Purifié 16S ADNr amplicons sont maintenant prêtes pour un certain nombre d&#39;analyses moléculaires qui permettent l&#39;identification taxinomique, tels que la construction de bibliothèques clone et le séquençage. </li></ol> 6. Les résultats représentatifs: Les résultats représentatifs sont décrits à l&#39;aide d&#39;une étude de putrescine bactéries dégradant comme un exemple. Des échantillons d&#39;eau ont été recueillies à partir d&#39;un site côtier de la Géorgie et traitées suivant les procédures décrites ci-dessus. Analyse FACS a révélé que plus de putrescine induit le développement d&#39;un groupe de bactéries à haute intensité de fluorescence FITC, indiquant un taux élevé de l&#39;incorporation de BrdU (Figure 1). Ces cellules ont été désignés comme étant à haut BrdU l&#39;incorporation des cellules (HIS) et devaient contiennent principalement putrescine bactéries dégradant. HIS sont manquants dans les commandes sans plus, qui ne contenait que des cellules avec des niveaux inférieurs de l&#39;incorporation de BrdU (LIS). Lis ont été s&#39;attend à ce que contiennent principalement bactérioplancton ont été incapables d&#39;utiliser la putrescine ajouté. HIS sont triés dans des tubes séparés et ensuite recueillis sur des filtres à membrane. Amplicons du gène 16S ARNr ont été obtenus pour les cellules en utilisant un filtre haute HI PCR. <img src="/files/ftp_upload/2855/2855fig1.jpg" alt="Figure 1"/> Figure 1. Cytométrie de flux sans plus de contrôle et de modèle composé modifiée (putrescine comme un exemple ici) échantillons prélevés après 24 h d&#39;incubation. Analyse de la distribution cellulaire a été basée sur (1) l&#39;intensité de fluorescence du FITC étiquetage (axe x), ce qui est positivement liée au niveau d&#39;incorporation de BrdU, et (2) la diffusion latérale (SSC, axe des y), ce qui est positivement liée à la cellule taille. Notations Gate est basée sur le niveau d&#39;incorporation de BrdU, (HI, haute-BrdU constitution; LI, faible BrdU constitution). Le pourcentage relatif de cellules HI et LI sont présentés dans les portes correspondantes.

<ol>
	<li>Pernthaler, A., Pernthaler, J., Schattenhofer, M., Amann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+DNA-synthesizing+bacterial+cells+in+coastal+North+Sea+plankton.">Identification of DNA-synthesizing bacterial cells in coastal North Sea plankton.</a> Appl. Environ. Microbiol. 68, 5728-5728 (2002).</li><li>Urbach, E., Vergin, K. L., Giovannoni, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunochemical+detection+and+isolation+of+DNA+from+metabolically+active+bacteria.">Immunochemical detection and isolation of DNA from metabolically active bacteria.</a> Appl. Environ. Microbiol. 65, 1207-12 (1999).</li><li>Mou, X. Z., Hodson, R. E., Moran, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterioplankton+assemblages+transforming+dissolved+organic+compounds+in+coastal+seawater.">Bacterioplankton assemblages transforming dissolved organic compounds in coastal seawater.</a> Environ. Microbiol. 9, 2025-2025 (2007).</li><li>Hodson, R. E., Dustman, W. A., Garg, R. P., Moran, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+PCR+for+visualization+of+microscale+distribution+of+specific+genes+and+gene+products+in+prokaryotic+communities.">In situ PCR for visualization of microscale distribution of specific genes and gene products in prokaryotic communities.</a> Appl. Environ. Microbiol. 61, 4074-4074 (1995).</li><li>Dinjens, W. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bromodeoxyuridine+(BrdU)+immunocytochemistry+by+exonuclease+III+(Exo+III)+digestion.">Bromodeoxyuridine (BrdU) immunocytochemistry by exonuclease III (Exo III) digestion.</a> Histochemistry. 98, 199-199 (1992).</li><li>Kirchman, D. L., Yu, L. Y., Fuchs, B. M., Amann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+bacterial+communities+in+aquatic+systems+as+revealed+by+filter+PCR.">Structure of bacterial communities in aquatic systems as revealed by filter PCR.</a> Aquat. Microb. Ecol. 26, 13-13 (2001).</li><li>Delong, E. F., Wickham, G. S., Pace, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phylogenetic+stains:+ribosomal+RNA-based+probes+for+the+identification+of+single+cells.">Phylogenetic stains: ribosomal RNA-based probes for the identification of single cells.</a> Science. 243, 1360-1360 (1989).</li><li>Artursson, V., Jansson, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+bromodeoxyuridine+immunocapture+to+identify+active+bacteria+associated+with+arbuscular+mycorrhizal+hyphae.">Use of bromodeoxyuridine immunocapture to identify active bacteria associated with arbuscular mycorrhizal hyphae.</a> Appl. Environ. Microbiol. 69, 6208-6208 (2003).</li><li>Mou, X. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+carbon+processing+by+generalist+species+in+the+coastal+ocean.">Bacterial carbon processing by generalist species in the coastal ocean.</a> Nature. 451, 708-708 (2008).</li><li>Mou, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow-cytometric+cell+sorting+and+subsequent+molecular+analyses+for+culture-independent+identification+of+bacterioplankton+involved+in+dimethylsulfoniopropionate+transformations.">Flow-cytometric cell sorting and subsequent molecular analyses for culture-independent identification of bacterioplankton involved in dimethylsulfoniopropionate transformations.</a> Appl. Environ. Microbiol. 71, 1405-1405 (2005).</li><li>Dean, F. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+human+genome+amplification+using+multiple+displacement+amplification.">Comprehensive human genome amplification using multiple displacement amplification.</a> Proc. Natl. Acad. Sci. U.S.A. 99, 5261-5261 (2002).</li></ol>

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Notre approche de couples BrdU constitution, FACS et 16S ADNr pour permettre l&#39;analyse des espèces au niveau d&#39;identification du bactérioplancton qui métabolisent les composants individuels du DOC dans les milieux aquatiques. Le dosage de l&#39;incorporation de BrdU étiquettes des cellules bactériennes en fonction des activités métaboliques, ce qui permet l&#39;analyse uniquement sur les bactéries actives et ne comprennent donc pas des cellules dormantes. Dans notre approche, l&#39;incorporation de BrdU ...

<table border="1"><tbody><tr><td> Nom </td><td> Tapez </td><td> Société </td><td> Le numéro de catalogue </td><td> Commentaires </td></tr><tr><td> BrdU </td><td> Réactifs </td><td> Sigma </td><td> B5002-5G </td><td></td></tr><tr><td> Le lysozyme </td><td> Réactifs </td><td> Sigma </td><td> L6876-5G </td><td></td></tr><tr><td> Protéinase K </td><td> Réactifs </td><td> Sigma </td><td> P2308-25MG </td><td></td></tr><tr><td> In Situ prolifération cellulaire Kit, fluos </td><td> Kit </td><td> Roche </td><td> 11810740001 </td><td> Consommer plus de l&#39;Anti-BrdU-fluos et le tampon d&#39;incubation par réaction que suggéré par le fabricant. </td></tr><tr><td> Chambres d&#39;incubation Cadre-Seal </td><td> Matériau </td><td> Bio-Rad </td><td> FSL-1201 </td><td></td></tr><tr><td> Polycarbonate filtres à membranes (142 mm de diamètre, 1,0 um-la taille des pores) </td><td> Matériau </td><td> Millipore </td><td> FALP14250 </td><td></td></tr><tr><td> Filtres à membrane en polycarbonate (25 mm de diamètre, de 0,2 um-la taille des pores) </td><td> Matériau </td><td> Millipore </td><td> FGLP02500 </td><td></td></tr><tr><td> illustra PuReTaq Ready-To-Go PCR Perles </td><td> Kit </td><td> GE Healthcare </td><td> 27-9559-01 </td><td></td></tr><tr><td> QIAquick gel extraction kit </td><td> Kit </td><td> QIAGEN </td><td> 28704 </td><td></td></tr><tr><td> FailSafe PCR System </td><td> Kit </td><td> EPICENTRE </td><td> FS99060 </td><td></td></tr></tbody></table>

bromodeoxyuridine (brdu) labeling and subsequent fluorescence activated cell sorting for culture-independent identification of dissolved organic carbon-degrading bacterioplankton

Les microbes sont les principaux agents de médiation de la dégradation de nombreuses carbone organique dissous (DOC) des substrats dans les milieux aquatiques. Toutefois, l&#39;identification des taxons bactériens qui transforment les piscines spécifiques de COD dans la nature pose un défi technique. Nous décrivons ici une approche qui couple bromodésoxyuridine (BrdU) l&#39;incorporation, tri cellulaire par fluorescence (FACS), et ARNr 16S basée sur l&#39;analyse moléculaire qui permet l&#39;identification indépendante de la culture du bactérioplancton capables de dégrader un composé DOC spécifiques dans les milieux aquatiques. Microcosmes bactérioplancton en triple sont mis en place pour recevoir les deux BrdU et un composé modèle de DOC (DOC amendements), ou seulement BrdU (sans ajout de contrôle). Substituts BrdU les positions de la thymidine dans l&#39;ADN bactérien nouvellement synthétisé et BrdU marqué l&#39;ADN peut être facilement immunodétectée 1,2. Grâce à une incubation de 24 h, bactérioplancton qui sont capables d&#39;utiliser le composé ajouté DOC devraient être activés sélectivement, et ont donc des niveaux plus élevés d&#39;incorporation de BrdU (cellules HI) que les non-réactive les cellules dans les modifications du DOC et les cellules dans un no- Outre les contrôles (faible incorporation de BrdU cellules, les cellules LI). Après immunodétection de fluorescence, les cellules HI sont distingués et physiquement séparé des cellules LI par tri cellulaire activé par fluorescence (FACS) 3. Tri DOC-réactive (cellules HI) sont extraits de l&#39;ADN et taxinomique identifiés par 16S ARNr ultérieure basée sur des analyses, y compris la PCR, la construction de bibliothèques clone et le séquençage.

Watch this Scientific Journal Video about Bromodeoxyuridine BrdU Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degradin

Bromodeoxyuridine BrdU Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degrading Bacterioplankton

Bactérioplancton environnement sont incubés avec un modèle de carbone organique dissous (DOC) composé et un réactif de marquage de l&#39;ADN, bromodésoxyuridine (BrdU). Ensuite, DOC-dégradants cellules sont séparées de la communauté en vrac en fonction de leur incorporation de BrdU élevée en utilisant la fluorescence tri cellulaire (FACS). Ces cellules sont ensuite identifiés par des analyses moléculaires ultérieures.

Bromodésoxyuridine (BrdU) l&#39;étiquetage et ultérieures de tri cellulaire activé par fluorescence de la culture indépendante Identification de carbone organique dissous dégradants bactérioplancton

Microbes are major agents mediating the degradation of numerous dissolved organic carbon (DOC) substrates in aquatic environments. However, identification ...

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Bromodeoxyuridine (BrdU) Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degrading Bacterioplankton

20.8K vues.  Kent State University. Bactérioplancton environnement sont incubés avec un modèle de carbone organique dissous (DOC) composé et un réactif de marquage de l'ADN, bromodésoxyuridine (BrdU). Ensuite, DOC-dégradants cellules sont séparées de la communauté en vrac en fonction de leur incorporation de BrdU élevée en utilisant la fluorescence tri cellulaire (FACS). Ces cellules sont ensuite identifiés par des analyses moléculaires ultérieures.

Vidéo: Bromodeoxyuridine BrdU Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degrading Bacterioplankton

Generation of Human CD40-activated B cells

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy 1-3. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) 3. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) 1,4. 
In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) 5. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). 
After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function 1, 4. CD40-B cells efficiently take up, process and present antigens to T cells 6. They do not only prime na&#970;ve, but also expand memory T cells 7,8. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors 9.

In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.

Génération de l&#39;homme CD40-B activés cellules

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

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Biologie

Fluorescence Activated Cell Sorting of Plant Protoplasts

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis.
An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: PSCR::GFP (endodermis and quiescent center) and PWOX5::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution.
FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time.
This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce cell types (e.g. quiescent center cells). Lastly, a growth setup for Arabidopsis seedlings is demonstrated that enables uncomplicated treatment of the plants prior to cell sorting (e.g. for the cell type-specific analysis of biotic or abiotic stress responses). Potential supplementary uses for FACS of plant protoplasts are discussed.

A method for isolating specific cell types from plant material is demonstrated. This technique employs transgenic marker lines expressing fluorescent proteins in particular cell types, cellular dissociation and Fluorescence Activated Cell Sorting. Additionally, a growth setup is established here that facilitates treatment of Arabidopsis thaliana seedlings prior to cell sorting.

Tri cellulaire activé par fluorescence des protoplastes végétaux

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental ...

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids.
 To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. 
 We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane. Additional lipid protein complexes can be identified using proteomics analysis of lipid binding protein co-purified with the lipid vesicles.

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

To test the interaction of a protein with its target lipid we used MACS and Annexin V-conjugated magnetic beads and lipid vesicles synthesized from the target lipid and Annexin V-binding phosphatidylserine. Proteins bound to the target lipid are co-purified and analyzed after elution from the beads.

Chromatographie d&#39;affinité lipidique des vésicules médiée utilisant magnétique tri cellulaire activé (LIMACS): une nouvelle méthode pour analyser les interactions protéines-lipides

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the Arabidopsis leaf is a complex organ made up of many different cell types and several structures. At the same time, the growing leaf contains cells at different stages of development, with the cells furthest from the petiole being the first to stop expanding and undergo senescence1. Different cells within the leaf are therefore dividing, elongating or differentiating; active, stressed or dead; and/or responding to stimuli in sub-sets of their cellular type at any one time. This makes genomic study of the leaf challenging: for example when analyzing expression data from whole leaves, signals from genetic networks operating in distinct cellular response zones or cell types will be confounded, resulting in an inaccurate profile being generated.
To address this, several methods have been described which enable studies of cell specific gene expression. These include laser-capture microdissection (LCM)2 or GFP expressing plants used for protoplast generation and subsequent fluorescence activated cell sorting (FACS)3,4, the recently described INTACT system for nuclear precipitation5 and immunoprecipitation of polysomes6.
FACS has been successfully used for a number of studies, including showing that the cell identity and distance from the root tip had a significant effect on the expression profiles of a large number of genes3,7. FACS of GFP lines have also been used to demonstrate cell-specific transcriptional regulation during root nitrogen responses and lateral root development8, salt stress9 auxin distribution in the root10 and to create a gene expression map of the Arabidopsis shoot apical meristem11. Although FACS has previously been used to sort Arabidopsis leaf derived protoplasts based on autofluorescence12,13, so far the use of FACS on Arabidopsis lines expressing GFP in the leaves has been very limited4. In the following protocol we describe a method for obtaining Arabidopsis leaf protoplasts that are compatible with FACS while minimizing the impact of the protoplast generation regime. We demonstrate the method using the KC464 Arabidopsis line, which express GFP in the adaxial epidermis14, the KC274 line, which express GFP in the vascular tissue14 and the TP382 Arabidopsis line, which express a double GFP construct linked to a nuclear localization signal in the guard cells (data not shown; Figure 2). We are currently using this method to study both cell-type specific expression during development and stress, as well as heterogeneous cell populations at various stages of senescence.

Cell Specific Analysis of Arabidopsis Leaves Using Fluorescence Activated Cell Sorting

A method for producing Arabidopsis leaf protoplasts that are compatible with fluorescence activated cell sorting (FACS), allowing for studies of specific cell populations. This method is compatible with any Arabidopsis line that expresses GFP in a subset of cells.

Analyse cellulaire spécifique de<em&gt; Arabidopsis</em&gt; Feuilles en utilisant la fluorescence par tri cellulaire activé

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves1. However, the ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

Optimisé méthodes de modélisation de coloration et de prolifération pour la surveillance division cellulaire en utilisant des colorants Suivi cellulaires

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting (FACS)

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their prominent presence is often associated with poor prognosis1,2. CAFs are an activated subpopulation of stromal fibroblasts, many of which express the myofibroblast marker &alpha;-SMA3. CAFs originate from local tissue fibroblasts as well as from bone marrow-derived cells recruited into the developing tumor and adopt a CAF phenotype under the influence of the tumor microenvironment4. CAFs were shown to facilitate tumor initiation, growth and progression through signaling that promotes tumor cell proliferation, angiogenesis, and invasion5-8. We demonstrated that CAFs enhance tumor growth by mediating tumor-promoting inflammation, starting at the earliest pre-neoplastic stages9. Despite increasing evidence of the key role CAFs play in facilitating tumor growth, studying CAFs has been an on-going challenge due to the lack of CAF-specific markers and the vast heterogeneity of these cells, with many subtypes co-existing in the tumor microenvironment10. Moreover, studying fibroblasts in vitro is hindered by the fact that their gene expression profile is often altered in tissue culture11,12 . To address this problem and to allow unbiased gene expression profiling of fibroblasts from fresh mouse and human tissues, we developed a method based on previous protocols for Fluorescence-Activated Cell Sorting (FACS)13,14. Our approach relies on utilizing PDGFR&alpha; as a surface marker to isolate fibroblasts from fresh mouse and human tissue. PDGFR&alpha; is abundantly expressed by both normal fibroblasts and CAFs9,15 . This method allows isolation of pure populations of normal fibroblasts and CAFs, including, but not restricted to &alpha;-SMA+ activated myofibroblasts. Isolated fibroblasts can then be used for characterization and comparison of the evolution of gene expression that occurs in CAFs during tumorigenesis. Indeed, we and others reported expression profiling of fibroblasts isolated by cell sorting16. This protocol was successfully performed to isolate and profile highly enriched populations of fibroblasts from skin, mammary, pancreas and lung tissues. Moreover, our method also allows culturing of sorted cells, in order to perform functional experiments and to avoid contamination by tumor cells, which is often a big obstacle when trying to culture CAFs.

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting FACS

Cancer Associated Fibroblasts (CAFs) facilitate tumor initiation, growth and progression through signaling that promotes proliferation, angiogenesis, and inflammation. Here we describe a method to isolate pure populations of normal fibroblasts and CAFs from fresh mouse and human tissues by cell sorting, using PDGFR&#x03B1; as a surface marker.

Isolation des fibroblastes normales et cancéreuses associées à partir de tissus frais par tri cellulaire activé par fluorescence (FACS)

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their ...

Nanogold Labeling of the Yeast Endosomal System for Ultrastructural Analyses

Endosomes are one of the major membrane sorting checkpoints in eukaryotic cells and they regulate recycling or destruction of proteins mostly from the plasma membrane and the Golgi. As a result the endosomal system plays a central role in maintaining cell homeostasis, and mutations in genes belonging to this network of organelles interconnected by vesicular transport, cause severe pathologies including cancer and neurobiological disorders. It is therefore of prime relevance to understand the mechanisms underlying the biogenesis and organization of the endosomal system. The yeast Saccharomyces cerevisiae has been pivotal in this task. To specifically label and analyze at the ultrastructural level the endosomal system of this model organism, we present here a detailed protocol for the positively charged nanogold uptake by spheroplasts followed by the visualization of these particles through a silver enhancement reaction. This method is also a valuable tool for the morphological examination of mutants with defects in endosomal trafficking. Moreover, it is not only applicable for ultrastructural examinations but it can also be combined with immunogold labelings for protein localization investigations.

Yeast, Saccharomyces cerevisiae, has been a key model organism to identify and study genes regulating the biogenesis and functions of the endosomal system. Here we present a detailed protocol for the specific labeling of the endosomal compartments for ultrastructural studies.

Nanogold étiquetage du système levure endosomique pour les analyses ultrastructurales

Endosomes are one of the major membrane sorting checkpoints in eukaryotic cells and they regulate recycling or destruction of proteins mostly from the ...

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific positions in DNA, RNA, proteins and small biomolecules. This natural methylation reaction can be expanded to a wide variety of alkylation reactions using synthetic cofactor analogues. Replacement of the reactive sulfonium center of AdoMet with an aziridine ring leads to cofactors which can be coupled with DNA by various DNA MTases. These aziridine cofactors can be equipped with reporter groups at different positions of the adenine moiety and used for Sequence-specific Methyltransferase-Induced Labeling of DNA (SMILing DNA). As a typical example we give a protocol for biotinylation of pBR322 plasmid DNA at the 5&#8217;-ATCGAT-3&#8217; sequence with the DNA MTase M.BseCI and the aziridine cofactor 6BAz in one step. Extension of the activated methyl group with unsaturated alkyl groups results in another class of AdoMet analogues which are used for methyltransferase-directed Transfer of Activated Groups (mTAG). Since the extended side chains are activated by the sulfonium center and the unsaturated bond, these cofactors are called double-activated AdoMet analogues. These analogues not only function as cofactors for DNA MTases, like the aziridine cofactors, but also for RNA, protein and small molecule MTases. They are typically used for enzymatic modification of MTase substrates with unique functional groups which are labeled with reporter groups in a second chemical step. This is exemplified in a protocol for fluorescence labeling of histone H3 protein. A small propargyl group is transferred from the cofactor analogue SeAdoYn to the protein by the histone H3 lysine 4 (H3K4) MTase Set7/9 followed by click labeling of the alkynylated histone H3 with TAMRA azide. MTase-mediated labeling with cofactor analogues is an enabling technology for many exciting applications including identification and functional study of MTase substrates as well as DNA genotyping and methylation detection.

DNA and proteins are sequence-specifically labeled with affinity or fluorescent reporter groups using DNA or protein methyltransferases and synthetic cofactor analogues. Depending on the cofactor specificity of the enzymes, aziridine or double activated cofactor analogues are employed for one- or two-step labeling.

Étiquetage spécifique de la séquence des acides nucléiques et des protéines avec Methyltransferases et le cofacteur Analogues

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific ...

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for cartilage tissue engineering. MSCs from synovial fluid (SF-MSCs) have been considered promising candidates for articular regeneration, and their potential therapeutic benefit has made them an important research topic of late. SF-MSCs from the knee cavity of the New Zealand white rabbit can be employed as an optimized translational model to assess human regenerative medicine. By means of CD90-based magnetic activated cell sorting (MACS) technologies, this protocol successfully obtains rabbit SF-MSCs (rbSF-MSCs) from this rabbit model and further fully demonstrates the MSC phenotype of these cells by inducing them to differentiate to osteoblasts, adipocytes, and chondrocytes. Therefore, this approach can be applied in cell biology research and tissue engineering using simple equipment and procedures.

This article presents a simple and economic protocol for the straightforward isolation and purification of mesenchymal stem cells from New Zealand white rabbit synovial fluid.

Cellule magnétique activé tri des stratégies visant à isoler et purifier Synovial fluide mésenchymateuses cellules souches dérivées d’un modèle de lapin

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for ...

Single Cell Micro-aspiration as an Alternative Strategy to Fluorescence-activated Cell Sorting for Giant Virus Mixture Separation

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or fluorescence activated cell sorting (FACS) applied to the viral population. However, when the viruses in the mixture have similar morphologic properties and one of the viruses multiplies slowly, the presence of two viruses is discovered at the stage of genome assembly and the viruses cannot be separated for further characterization. To solve this problem, we developed a single cell micro-aspiration procedure that allows for separation and cloning of highly similar viruses. In the present work, we present how this alternative strategy allowed us to separate the small viral subpopulations of Clandestinovirus ST1 and Usurpativirus LCD7, giant viruses that grow slowly and do not lead to amoebal lysis compared to the lytic and fast-growing Faustovirus. Purity control was assessed by specific gene amplification and viruses were produced for further characterization.

Here, we describe a single cell micro-aspiration method for the separation of infected amoebae. In order to separate viral subpopulations in Vermamoeba vermiformis infected by Faustoviruses and unknown giant viruses, we developed the protocol detailed below and demonstrated its ability to separate two low-abundance novel giant viruses.

Micro-aspiration à cellule unique comme stratégie alternative au tri cellulaire activé par fluorescence pour la séparation du mélange de virus géants

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or ...