Il finanziamento di questo progetto è stato fornito dal National Science Foundation concede OCE1029607 (a XM) e MCB0702125 (a MAM) e Gordon and Betty Moore Foundation (a MAM).

1. Acqua esempio per l&#39;elaborazione <ol><li> Filtrare l&#39;acqua 10L ambientale attraverso 1 micron-pori di dimensioni filtri a membrana per rimuovere le particelle più grandi e bacteriovores. Raccogliere il filtrato l&#39;acqua in una damigiana. </li><li> Trasferire 36 ml ciascuno filtrato in 3 provette sterili Eppendorf (50 ml) contenente 4 ml di soluzione appena preparata paraformaldeide (PFA; 10%). Incubare per 2 ore a temperatura ambiente per preservare le cellule. Raccogliere le cellule su 0,22-micron-pori di dimensioni filtri a membrana bianca per filtrazione a vuoto. Lavare il filtro facendo passare 10 ml di soluzione salina tampone fosfato (PBS) attraverso il filtro per filtrazione a vuoto. Etichetta i filtri come controlli negativi e conservarli a -20 ° C. </li></ol> Passi 1,3-1,4 sono opzionali per stabilire DOC-limitata condizioni. <ol start="3"><li> Aggiungere una miscela di azoto inorganico e fosforo (5 mM NH 4 Cl, 5 mM NaNO 3 e 1μM NaH 2 PO 4 concentrazione finale) nella damigiana. </li><li> Incubare al buio a temperatura in situ per 48 ore con agitazioni occasionali. </li></ol> 2. Microcosmo creazione e l&#39;incubazione <ol><li> Riempire ogni sei 1-L vasi in vetro con 800 ml di acqua campione dalla damigiana di passo da 1,4 a stabilire microcosmi. Aggiungi BrdU (10 micron, concentrazione finale) per ogni microcosmo. Mescolare bene. </li><li> Aggiungere 1 ml di modello DOC soluzione composta in tre dei microcosmi, queste modifiche serviranno come DOC. Aggiungere 1 ml di PBS sterile nei tre microcosmi restanti, queste serviranno come no-Oltre controlli. </li><li> Incubare tutte le microcosmi in un agitatore incubatore e incubare al buio a temperatura in situ agitando a 100 giri al minuto. </li><li> Raccogliere 36 ml di campione d&#39;acqua da ogni microcosmo e trasferire 50 ml in provette sterili Eppendorf in momenti di 0, 8, 16 e 24 ore. Immediatamente aggiungere 4 ml di fresca PFA (10%) ai tubi di raccolta e incubare per 2 ore a temperatura ambiente per preservare le cellule. </li><li> Filtro conservate cellule attraverso 0.22-micron-pori di dimensioni filtri a membrana in policarbonato. Lavare i filtri con 10 ml di PBS. Procedere immediatamente alla fase successiva o conservare i filtri a -20 ° C. </li></ol> 3. Immunolocalizzazione in situ per incorporazione di BrdU <ol><li> Scongelare i filtri (campioni DOC modificato, senza aggiunta controlli e controlli negativi) a temperatura ambiente. </li><li> Applicare 1 ml di soluzione di lisozima [10 mg / ml di lisozima albume in 100 mM Tris, 50 nM EDTA (pH = 8)] 4 per coprire le cellule batteriche sul filtro. Incubare a temperatura ambiente per 30 minuti. Lavare il filtro facendo passare 10 ml di PBS attraverso di essa sotto aspirazione. </li><li> Aggiungere 1 ml di soluzione di proteinasi K [2 mg / ml di proteinasi K in 100 mM Tris, 50 nM EDTA (pH = 8)] 4 per coprire le cellule batteriche sul filtro. Incubare a temperatura ambiente per 30 minuti. Lavare il filtro facendo passare 10 ml di PBS attraverso di essa sotto aspirazione. </li><li> Aggiungere 1 ml di soluzione esonucleasi [esonucleasi III (50 U / ml) in 5 mM MgCl 2 e 50 mM Tris-HCl] 5 per coprire le cellule batteriche sul filtro. Incubare a 37 ° C per 30 minuti. Lavare il filtro facendo passare 10 ml di PBS attraverso di essa sotto aspirazione. </li><li> Montare frame-sigillo camere di incubazione (Bio-Rad) secondo le istruzioni del produttore. </li><li> Tagliate il filtro in ottavi con una lama sterile su un alcool-superficie asciutta e pulita. </li><li> Utilizzando pinze, posto una sezione ottavo di filtro montato in un quadro di tenuta della camera di incubazione (otto frame-sigillo camere sono necessari per ogni campione filtro). Il lato della sezione di filtro che contiene cellule deve essere rivolto verso l&#39;alto. L&#39;umidità nella parte posteriore del filtro permetterà il bastone filtro alla diapositiva. Se il filtro si secchi, applicare una piccola goccia (2 mL) di DIH 2 O nel centro della camera prima di mettere la sezione filtro sul vetrino. </li></ol> Passi 3,8-3,20 utilizzare i reagenti dalla ROCHE In Situ Kit proliferazione cellulare, FLUOS seguenti procedure modificate dalle istruzioni del produttore. Fatta eccezione per la PBS, tutti i reagenti sono forniti nel kit. <ol start="8"><li> Applicare tampone abbastanza incubazione (0,5% di siero bovino albume, 0,1% Tween20 in PBS,) per coprire in modo uniforme l&#39;intera sezione del filtro nella camera di incubazione senza causare overflow di una volta la guarnizione viene applicata. </li><li> Inserire una struttura di copertura in poliestere sopra la struttura della camera di incubazione. Premere verso il basso per sigillare ermeticamente la camera di incubazione. Evitare bolle d&#39;aria al di sopra del filtro. Incubare le camere sigillate per 10 minuti a temperatura ambiente al buio. </li><li> Rimuovere il sigillo di poliestere e aprire le camere di incubazione. (Nota: l&#39;apertura delle camere a volte tirerà su il sigillo telaio dalla diapositiva In questo caso, preparare una nuova camera per i seguenti passaggi..) </li><li> Pipettare il buffer di incubazione da un angolo della camera. Evitare il contatto con il filtro. </li><li> Lavare il filtro pipettando gentilmente 100 l di PBS efuori dalla camera di 3 volte. </li><li> Preparare anti-BrdU-FLUOS soluzione di lavoro seguendo la procedura raccomandata dal produttore immediatamente prima dell&#39;uso. </li><li> Pipettare 120 microlitri anti-BrdU-FLUOS soluzione di lavoro sul filtro, avendo cura di coprire uniformemente tutta la superficie. </li><li> Richiudere la camera con una copertura in poliestere nuovo. Evitare bolle d&#39;aria sulla parte superiore del filtro. Incubare la camera al buio a 37 ° C per 3 ore. Questo passo etichetta BrdU DNA incorporato in situ con isotiocianato di fluorescina (FITC). </li><li> Rimuovere la copertura in poliestere e aprire la camera di incubazione. Pipettare il anti-BrdU-FLUOS soluzione di lavoro. Lavare il filtro 3 volte con PBS. </li><li> Trasferire il filtro dalla camera di incubazione per una superficie sterile. Tagliate la sezione filtro in piccoli pezzi con una lama sterile. </li><li> Trasferire il filtro in 2 pezzi microprovette ml, contenenti ciascuno 1 ml di PBS. Strettamente tappo del tubo e sigillare con parafilm. Incubare a 37 ° C e 200 rpm per 10 min. </li><li> Fissare i tubi in un vortice vortex e alla velocità massima per 5 minuti. Pipetta il sopranatante in una provetta sterile 15 ml Eppendorf. Ripetere le fasi di incubazione e vortex per 5 volte di più. Combina il surnatante nello stesso tubo 15 ml Eppendorf per ogni campione. Tipicamente, l&#39;80% di cellule possono essere recuperati in sospensione. </li><li> Conservare il supernatante con le cellule risospese a 4 ° C. Ordina entro 2 giorni. </li></ol> 4. FACS analisi Una procedura per un citometro di flusso BD FACSAria e relativo software è descritto qui. <ol><li> Ottimizzare l&#39;impostazione delle fasi del citometro a flusso seguito consigliato dal produttore. Ciò comporta: regolando il controllo di amplificazione per impostare i parametri richiesti e breakoff lo sweet spot, ottimizzando il ritardo laser e fattori di scala area per la pressione guaina esperimento e ottimizzare le impostazioni per FSC e tensioni SSC, soglia FSC, FSC scala fluorescenza, fluorescenza tensioni PMT , ecc </li><li> Esegui campioni di controllo negativo sul citometro a flusso (FCM) sulla base di intensità di fluorescenza di dispersione FITC e laterali (SSC). Aumentare la soglia fino a quando non FITC cellule nei controlli negativi possono essere visualizzati dal FITC-SSC visualizzazione acquisizione. </li><li> Eseguire senza aggiunta campioni di controllo e esaminare il modello di distribuzione di 10.000 celle in base a FITC e acquisizione SSC. Definire un cancello di racchiudere tutte le cellule e designare come &quot;celle a bassa intensità&quot; (LIS). </li><li> Esegui DOC-modificato campioni ed esaminare il modello di distribuzione di 10.000 celle in base a FITC e SSC. Alcune cellule apparirà nel cancello preset LI. Definire un altro cancello per racchiudere le cellule che hanno una maggiore intensità di fluorescenza (HIS) di LIS. Acquisire le statistiche per vedere la relativa abbondanza di cellule gated. </li><li> Celle di tipo HI in provette contenenti 500 microlitri PBS a &quot;purificare 1 goccia&quot; modalità. Termina ordinamento quando il numero dei suoi raggiunge 500.000 conta. </li></ol> 5. Filtro amplificazione di geni rRNA 16S Filtro procedure di PCR vengono modificati da Kirchman et al 6. <ol><li> Filtro cellule ordinati su un bianco, 0,22 micron-pori di dimensioni, 25 mm di diametro, in policarbonato membrana del filtro. Tagliare il bordo del filtro che contiene le cellule batteriche con una lama sterile. Tagliate il filtro in 8 pezzi uguali dimensioni. </li><li> Posizionare un pezzo unico filtro in un tubo di reazione di PCR, con le cellule rivolti verso l&#39;interno del tubo. Aggiungere 45 microlitri di acqua grado di PCR nel tubo di reazione PCR. Immergere il filtro completamente in acqua. </li><li> Aggiungere 2 ROCHE illustrazioni PuReTaq Read-To-Go PCR Beads nel tubo di reazione PCR, mescolare brevemente nel vortex. Aggiungere 2 microlitri ciascuno degli avanti e indietro primer gene rRNA 16S (0,4 microM concentrazione finale per ciascun primer), come 27F e 1492R 7, in ciascuna delle provette di reazione della PCR. </li><li> (Facoltativo) Aggiungere 1ml soluzione di albumina sierica bovina (BSA, concentrazione finale è 30μg/100 microlitri) alla miscela di reazione PCR per aiutare inibitori dell&#39;amplificazione assorbire. Se uno sceglie di non aggiungere BSA, aggiungere 1ml di acqua, invece. </li><li> Eseguire la PCR su un termociclatore. Un tocco giù programma PCR si raccomanda, che è la temperatura di ricottura sequenzialmente decrescenti 62-52 ° C di 1 ° C per ciclo per 11 cicli, seguiti da 15 cicli con temperatura di annealing di 52 ° C. Tutti i cicli sono denaturazione (a 95 ° C), ricottura (a 62 a 52 ° C), ed estensione (a 72 ° C) le misure di durata 50 anni. Un primo 3-min di denaturazione e una finale di 10 minuti passo estensione è inoltre inclusa nel programma PCR. </li><li> Conferma PCR mediante elettroforesi su un bromuro macchiato etidio 1% gel. Dell&#39;accisa gli ampliconi della PCR dal gel e pulire con l&#39;estrazione QIAGEN QIAquick kit gel. </li><li> Eseguire due ulteriori amplificazioni PCR per ogni campione, ogni volta che utilizzano una nuova sezione filtro. Dopo la PCR gel purificatiin poi, la piscina ampliconi del campione stesso insieme. Purificata 16S rDNA ampliconi sono ora pronti per una serie di analisi molecolari che permettono l&#39;identificazione tassonomica, come la costruzione clone biblioteca e sequenziamento. </li></ol> 6. Rappresentante dei risultati: I risultati rappresentativi sono descritte utilizzando uno studio di putrescina-degradante batteri come esempio. Campioni di acqua sono stati raccolti in un sito costiero della Georgia e trattati seguendo le procedure sopra descritte. FACS analisi ha rivelato che oltre putrescina indotto lo sviluppo di un gruppo di batteri ad alta intensità FITC fluorescenza, indicando alto tasso di incorporazione di BrdU (Figura 1). Queste cellule sono state designate come high-BrdU-incorporazione cellule (HIS) e ci si aspettava che contengono principalmente putrescina-degradanti batteri. Il suo mancavano nel no-aggiunta controlli, che conteneva solo le cellule con livelli inferiori di incorporazione di BrdU (LIS). LI ci si aspettava che contengono principalmente batterioplancton che sono stati in grado di utilizzare putrescina aggiunto. I suoi sono stati ordinati in tubi separati e poi raccolto su filtri a membrana. Ampliconi gene 16S rRNA sono stati ottenuti per le celle ad alta HI con filtro PCR. <img src="/files/ftp_upload/2855/2855fig1.jpg" alt="Figura 1"/> Figura 1. Analisi citofluorimetrica la mancata aggiunta di controllo e il modello-compound-modificato (putrescina ad esempio qui) i campioni raccolti dopo 24 ore di incubazione. Analisi della distribuzione delle cellule si è basata su (1) intensità di fluorescenza del FITC etichettatura (asse x), che è positivamente correlata al livello di incorporazione di BrdU, e (2) side scatter (SSC, asse y), che è positivamente correlato alla cella dimensioni. Notazione cancello si basa sul livello di incorporazione di BrdU, (HI, high-BrdU-incorporazione, LI, basso BrdU-incorporazione). La percentuale relativa di HI e cellule LI sono riportati nella cancelli corrispondente.

<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>

Nessun conflitto di interessi dichiarati.

Il nostro approccio coppie BrdU incorporazione, FACS analisi e 16S rDNA per consentire l&#39;identificazione delle specie a livello di batterioplancton che metabolizzano i singoli componenti DOC in ambienti acquatici. Il saggio di incorporazione di BrdU etichette cellule batteriche sulla base di attività metabolica, che permette l&#39;analisi solo sui batteri attivi e quindi non include le cellule dormienti. Nel nostro approccio, incorporazione di BrdU è nei batteri in situ immunodetected e che hanno...

<table border="1"><tbody><tr><td> Nome </td><td> Tipo </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td> BrdU </td><td> Reagente </td><td> Sigma </td><td> B5002-5G </td><td></td></tr><tr><td> Lisozima </td><td> Reagente </td><td> Sigma </td><td> L6876-5G </td><td></td></tr><tr><td> Proteinasi K </td><td> Reagente </td><td> Sigma </td><td> P2308-25mg </td><td></td></tr><tr><td> In Situ Kit proliferazione delle cellule, FLUOS </td><td> Kit </td><td> Roche </td><td> 11810740001 </td><td> Consumano più di Anti-BrdU-FLUOS e buffer di incubazione per reazione rispetto a quanto suggerito dal produttore. </td></tr><tr><td> Frame-Seal incubazione Chambers </td><td> Materiale </td><td> Bio-Rad </td><td> SLF-1201 </td><td></td></tr><tr><td> I filtri a membrana in policarbonato (142 mm di diametro, 1,0 micron-poro-size) </td><td> Materiale </td><td> Millipore </td><td> FALP14250 </td><td></td></tr><tr><td> I filtri a membrana in policarbonato (25-mm di diametro, 0,2 micron a poro-size) </td><td> Materiale </td><td> Millipore </td><td> FGLP02500 </td><td></td></tr><tr><td> illustrazioni PuReTaq Ready-To-Go PCR Beads </td><td> Kit </td><td> GE Healthcare </td><td> 27-9559-01 </td><td></td></tr><tr><td> QIAquick gel kit di estrazione </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

I microbi sono agenti principali mediare la degradazione di numerosi carbonio organico disciolto (DOC) substrati in ambienti acquatici. Tuttavia, l&#39;identificazione di taxa batterici che trasformano piscine specifiche di DOC in natura rappresenta una sfida tecnica. Qui si descrive un approccio che le coppie bromodeossiuridina (BrdU) l&#39;incorporazione, fluorescenza cell sorting attivato (FACS) e 16S rRNA gene-based analisi molecolare che consente di cultura indipendente identificazione di batterioplancton in grado di degradare un composto DOC specifico in ambienti acquatici. Microcosmi batterioplancton tre esemplari sono impostati per ricevere sia BrdU e un composto modello DOC (DOC emendamenti), oppure solo BrdU (senza aggiunta di controllo). Sostituti BrdU le posizioni di timidina nel DNA di nuova sintesi batterica e BrdU marcato DNA può essere facilmente immunodetected 1,2. Attraverso una 24-ore di incubazione, batterioplancton che sono in grado di utilizzare il composto aggiunto DOC dovrebbero essere selettivamente attivato, e quindi hanno più alti livelli di incorporazione di BrdU (cellule HI) che non rispondono le cellule negli emendamenti DOC e le cellule nel no- Inoltre controlla (basso cellule incorporazione di BrdU, le cellule LI). Dopo immunolocalizzazione fluorescenza, le cellule si distinguono HI e fisicamente separato dalle cellule LI da fluorescenza separazione delle cellule attivate (FACS) 3. Ordinati DOC-responsive cellule (cellule HI) vengono estratte per il DNA e tassonomicamente individuate attraverso successive rRNA 16S a base genetica tra cui analisi di PCR, la costruzione della libreria clone e sequenziamento.

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

Batterioplancton ambientali vengono incubate con un modello di carbonio organico disciolto (DOC) e un reattivo composto di etichettatura del DNA, bromodeossiuridina (BrdU). In seguito, DOC-degradante celle sono separate dalla comunità di massa in base alla loro elevata incorporazione di BrdU usando fluorescenza separazione delle cellule attivate (FACS). Queste cellule vengono poi identificati da successive analisi molecolari.

Bromodeossiuridina (BrdU) Etichettatura e successive ordinamento fluorescenza cellulare attivo per la cultura indipendente Identificazione di carbonio organico disciolto-degradanti batterioplancton

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

bromodeoxyuridine-brdu-labeling-subsequent-fluorescence-activated

Research

JoVE Journal

Biology

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

20.8K Views.  Kent State University. Batterioplancton ambientali vengono incubate con un modello di carbonio organico disciolto (DOC) e un reattivo composto di etichettatura del DNA, bromodeossiuridina (BrdU). In seguito, DOC-degradante celle sono separate dalla comunità di massa in base alla loro elevata incorporazione di BrdU usando fluorescenza separazione delle cellule attivate (FACS). Queste cellule vengono poi identificati da successive analisi molecolari.

Video: 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.

Generazione di Human CD40 delle cellule B attivate

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

Ricerca

Biologia

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.

Fluorescenza separazione delle cellule attivate delle protoplasti impianto

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.

Vescicole lipidiche-mediata Cromatografia di affinità mediante Magnetic separazione delle cellule attivate (LIMACS): un nuovo metodo per l&#39;analisi delle interazioni proteina-lipide

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.

Cella di analisi specifica di<em&gt; Arabidopsis</em&gt; Foglie usando fluorescenza activated cell sorting

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.

Ottimizzato Metodi di modellazione e colorazione proliferazione per il monitoraggio divisione cellulare utilizzando coloranti Tracking Cellulari

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.

Isolamento di fibroblasti normali e tumorali associata da tessuti freschi di fluorescenza 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 ...

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 Etichettatura del Sistema lievito endosomiale per le analisi ultrastrutturali

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.

Specifici Sequenza Etichettatura degli acidi nucleici e proteine ​​con metiltransferasi e cofattore Analoghi

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.

Magnetico-Activated Cell Sorting strategie per isolare e purificare Synovial cellule staminali mesenchimali liquido derivato da un modello di coniglio

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-aspirazione a cella singola come strategia alternativa per lo smistamento cellulare attivato dalla fluorescenza per la separazione delle miscele di virus giganti

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 ...