Ringraziamo il Dott. Yitzchak Oschry e il Dr. Orit Sagi-Asif per il loro aiuto con la selezione FACS. Questa ricerca è stata sostenuta da sovvenzioni a NE del programma europeo per il Settimo programma quadro dell&#39;Unione (FP7/2007-2013) nell&#39;ambito della convenzione di sovvenzione n ° [276890], da Israele Cancer Association (# 20110078), e da Israele Cancer Research Fund (Ricerca Career Development Award).

1. Dissezione tessuto mammario o con la pelle di topi <ol><li> Prima di sezionare il tessuto desiderato dai topi, preparare le seguenti forniture e dei reagenti: Materiali di consumo: <ul><li> Strumenti chirurgici (lavato con detergente e poi immersi in 70% etanolo). </li><li> Superficie di polistirolo per appoggiare il mouse su durante la dissezione. </li><li> Pins o aghi per tenere i topi durante la dissezione. </li><li> Vaso di vetro con coperchio per la digestione, contenente barra di agitazione magnetica (autoclave). </li><li> Bagnomaria a 37 ° C, contenente un agitatore magnetico sommergibile e acqua sufficiente per immergere il vaso digestione, mentre appoggio alla piastra di agitazione. </li></ul> Reagenti: <ul><li> FACS buffer di I: (4 ° C): fosfato Dulbecco Buffered Saline CMF (Calcio Magnesio gratuita) + 0,5% di BSA </li><li> Soluzione di collagenasi (immediatamente prima dell&#39;uso): 0,05 g Collagenasi II 0,05 g Collagenasi IV 0,01 g Deoxyribonuclease 20ml FACS tampone I. Tenere soluzione di collagenasi in ghiaccio fino al momento dell&#39;uso. </li><li> PharmLyse (cloruro di ammonio Reagente di lisi). </li><li> FACS tampone II: fosfato Dulbecco Buffered Saline CMF + 1% FCS. </li></ul></li><li> Dissezione delle ghiandole mammarie (vedi anche 17). <ul><li> Eutanasia topi di sesso femminile con anidride carbonica (CO 2) inalazione. </li><li> Su una superficie di polistirolo, posizionare il mouse sul dorso e pin saldamente tutti e quattro gli arti con aghi calibro 25 (Figura 1A). Spruzzare il mouse generosamente con il 70% di etanolo. </li><li> Utilizzando pinze, tirare la pelle addominale sulla linea mediana e fare una piccola incisione con le forbici affilate. </li><li> Partendo dall&#39;incisione, tagliare la pelle fino al collo dell&#39;animale evitando pungere cavità addominale o toracica. </li><li> Tagliare la pelle delle gambe posteriori della incisione mediana verso la gamba, risultanti in una &quot;forma Y&quot;. </li><li> Tirare la pelle lontano dal corpo del mouse e definire, esponendole ghiandole mammarie fissato al lato inferiore della pelle (Figura 1B). </li><li> Utilizzare Q-tips per separare delicatamente ghiandole mammarie dalla pelle durante il taglio di tessuto connettivo, con piccole forbici affilate per separare la ghiandola mammaria verso la colonna vertebrale del mouse. </li><li> Le ghiandole addominali (# 4, 5) sono i migliori per questo protocollo. È inoltre possibile utilizzare la ghiandola mammaria 2 °, ma tenere a mente che queste ghiandole sono anatomicamente trova in prossimità del muscolo pettorale, che può essere difficile da separare, con conseguente cellule di origine del tessuto misto. </li><li> Nota: Quando la dissezione del tessuto tumorale, si consiglia di rimuovere le aree necrotiche. </li></ul></li><li> La dissezione del tessuto cutaneo: Il modo più semplice per ottenere tessuto cutaneo, senza avere a che fare con la rimozione dei capelli è quello di utilizzare le orecchie del mouse. Se una maggiore quantità di tessuto è necessario, è possibile radersi i capelli dal tronco del mouse e sezionare la pelle. <ul><li> Eutanasia topo, utilizzando CO 2 inalazione. </li><li> Utilizzando forbici affilate, tagliare entrambe le orecchie e mettere subito in vaso di digestione contenente un piccolo volume (~ 0,5 ml) di PBS su ghiaccio. </li></ul></li></ol> 2. Preparazione della ghiandola mammaria / pelle Sospensione singola cella <ul><li> Posizionare ghiandole mammarie / pelle in vaso di digestione contenente un piccolo volume di PBS sul ghiaccio. Se il tessuto è sanguinosa x2 lavaggio con PBS, e quindi eliminare l&#39;eccesso di PBS. </li><li> Tritare accuratamente con le forbici curve. </li><li> Aggiungi (nota: questa quantità di soluzione di collagenasi in modo efficiente dissociare circa 5 g di tessuto mammario o tumore e le orecchie da fino a 15 topi) 20 ml di soluzione di collagenasi. </li><li> Mettere immediatamente mescolare piastra a 37 ° C in bagno d&#39;acqua, e incubare per 15 minuti sotto agitazione al tasso medio. </li></ul> Nota: i tempi ottimali di incubazione può variare se digerire altri tessuti. <ul><li> Per arrestare la reazione, aggiungere 30 ml DMEM freddo + 10% FCS. </li><li> Filtrare il tessuto / media throug miscela70 ha filtro cella um collocato su un tubo da 50 ml. </li><li> Centrifugare la provetta conica per 5 min a 450 xg a 4 ° C. </li></ul> 3. Blood Red Cells Lysis Nota: questo passaggio non è necessario se i topi sono stati il cuore perfusi con PBS prima di essere sacrificati. <ul><li> Aspirare il supernatante usando una pipetta di vetro collegato a vuoto, senza disturbare il pellet. </li><li> Per lisare RBC, risospendere il pellet cellulare in 10 ml di soluzione di PharmLyse e incubare per 5 minuti a temperatura ambiente. </li><li> Neutralizzare la soluzione PharmLyse con l&#39;aggiunta di circa 40 ml di FACS tampone I. </li><li> Centrifugare la provetta conica con le cellule per 5 min a 450 xg a 4 ° C. </li><li> Aspirare il surnatante. </li></ul> 4. Blocco di Fc endogena <ul><li> Opzionale: cellule Count per determinare il volume appropriato per le fasi successive. </li><li> Risospendere le cellule in 1-3 ml di FACS buffer di I (a seconda on numero di cellule, e la quantità di anticorpi che si desidera utilizzare). </li><li> Aggiungi FcBlock (anti-mouse CD16/CD32) per raggiungere diluizione 1:50 (10 pg / ml), e incubare su ghiaccio per 10-20 &#39;. Non c&#39;è bisogno di lavare FcBlock. </li></ul> 5. Colorazione <ul><li> Estrarre aliquote di 100 pl per provette Eppendorf da utilizzare come un non-tinto controllo e comandi a singolo colore (a seconda del numero di anticorpi fluorescenti utilizzate). </li><li> Per fibroblasti etichetta: aggiungere anti-PDGFRα (direttamente coniugato fluoroforo) ad una diluizione di 1:50 (10 pg / ml). </li><li> Per etichettare popolazioni cellulari (es. cellule immunitarie, macrofagi, cellule endoteliali ecc): aggiungere appropriati anticorpi fluorescenti coniugati a marcatori di superficie, ad una diluizione 1:50. </li></ul> Nota: sebbene PDGFRα è un marcatore robusto per fibroblasti, si raccomanda di includere anticorpi per cellule immunitarie e cellule epiteliali in modo da escludere qualsiasi positivo doppiapopolazioni e quindi aumentare la purezza dei fibroblasti isolati. <ul><li> Aggiungere 2 microlitri di ciascun anticorpo a ciascuno dei singoli tubi eppendorf colore controllo. </li><li> Incubare le cellule con anticorpi per 1 ora su ghiaccio al buio. </li><li> Per lavare: riempire i tubi con tampone FACS I e girare per 5 min a 450 xg a 4 ° C. </li><li> Aspirare il surnatante, risospendere le cellule in tampone FACS I a una concentrazione di circa 2 x 10 6 cellule / ml, o qualsiasi concentrazione più appropriato per l&#39;ordinamento con il sorter disponibili FACS. </li></ul> Opzionale: Le cellule possono essere risospese in tampone FACS II per la durata di smistamento. Questo può migliorare la vitalità cellulare. Tuttavia, l&#39;esposizione a fattori nel siero possono avere un effetto espressione genica, che deve essere testato, e prese in considerazione. <ul><li> Trasferire cellule marcate in tubi FACS con filtro migliori: celle filtranti nei tubi per evitare grumi di cellule. </li><li> Per escludere cellule morte, aggiungere 7AAD (0,5 mcg / ml) o Propidium Ioduro (PI) ai campioni (tranne che per il non-macchiato di controllo). </li></ul> Nota: Per evitare di sprecare le celle, è possibile aggiungere 7AAD/PI al non-tinto campione dopo la registrazione nel FACS, e usare di nuovo come 7AAD-solo il controllo. <ul><li> Conservare i campioni in ghiaccio durante l&#39;analisi. </li></ul> 6. Isolamento delle cellule da FACS (Nota: questa parte del protocollo richiede conoscenza nella gestione di un selezionatore FACS, ad esempio BD FACS AriaII, o l&#39;assistenza di un tecnico specializzato). <ul><li> Utilizzando il software di FACS sorter (BD FACSDiva), preparare grafici di analisi adeguati per l&#39;analisi forward scatter (FSC), dispersione laterale (SSC), 7AAD, FITC, PE e qualsiasi altro fluoroforo utilizzato per etichettare le cellule (Figura 2). </li><li> Prima di caricare ogni campione al cell sorter, vortice brevemente per risospendere le cellule. </li><li> Inizia analizzando il campione non colorato per impostare il gating della popolazione cellulare totale, per escludere il celL detriti, e per determinare autofluorescenza o colorazione di fondo. </li><li> Analizzare campione senza macchia + 7AAD per determinare e porta la popolazione di cellule vive della popolazione cellulare totale. </li><li> Analizzare ogni singolo controllo del colore per determinare e calibrare i parametri quali la compensazione. </li><li> Analizzare il campione colorato per definire la posizione di porte per l&#39;ordinamento. </li><li> Una volta che i parametri e le porte per le popolazioni cellulari desiderati sono stati impostati, iniziare l&#39;ordinamento dei popolazioni di cellule etichettate in provette Eppendorf o provette da 15 ml coniche contenenti terreno di coltura o tampone di lisi RNA (come RLT tampone o Trizol). </li></ul> Nota: Se l&#39;ordinamento di un gran numero di cellule, non è consigliabile per ordinare direttamente nel tampone di lisi RNA, come il volume di liquido guaina accompagna le cellule diluire il tampone di lisi e ridurre la resa. <ul><li> Spin celle in basso subito dopo la cernita è finito, e il trasferimento in terreno fresco o tampone di lisi RNA, dein attesa dello scopo dell&#39;esperimento. </li><li> Se l&#39;ordinamento, al fine di isolate cellule di coltura, eseguire sorta sterile. Per migliore recupero delle cellule, utilizzare rosso fenolo libero DMEM + 5% FCS anziché FACS tampone II e raccogliere cellule in mezzo di coltura con 20% FCS. </li><li> Se la coltura ordinati fibroblasti, alwaysplate su piastre rivestite di collagene e mai direttamente sulla plastica come questo si traduce in attivazione dei fibroblasti che portano a cambiamenti nella loro espressione genica. </li></ul>

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Nessun conflitto di interessi dichiarati.

Mentre esperimenti condotti in coltura tissutale può essere informativo e suggerire principi funzionali che possono essere verificati in vivo, è noto che si verificano grandi cambiamenti nell&#39;espressione genica di cellule in coltura 11,12. Per evitare un passo coltura tissutale a profilare espressione genica in fibroblasti, abbiamo sviluppato un protocollo che permette di isolare normali, così come cancro-associate fibroblasti di topo freschi o tessuti umani. Questo protocollo è stato applica...

<table border="1"&gt;<tbody&gt;<tr&gt;<td<strong&gt; Nome del reagente</strong</td&gt;<td<strong&gt; Azienda</strong</td&gt;<td<strong&gt; Numero di catalogo</strong</td&gt;<td<strong&gt; Commenti (opzionale)</strong</td&gt;</tr&gt;<tr&gt;<td&gt; DMEM</td&gt;<td&gt; Gibco</td&gt;<td&gt; 41965</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; PBS</td&gt;<td&gt; Biologia Industries</td&gt;<td&gt; 02-023-1A</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Collagenasi II</td&gt;<td&gt; Worthington</td&gt;<td&gt; LS4176</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Collagenasi IV</td&gt;<td&gt; Worthington</td&gt;<td&gt; LS4188</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Deoxyribonuclease</td&gt;<td&gt; Worthington</td&gt;<td&gt; LS2007</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; PharmLyse</td&gt;<td&gt; BD</td&gt;<td&gt; 555899</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Filtro cella 70 pm</td&gt;<td&gt; SPL</td&gt;<td&gt; 93070</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Purificato anti-topo CD16/CD32</td&gt;<td&gt; BD Pharmingen</td&gt;<td&gt; 553142</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Via sonda (7AAD)</td&gt;<td&gt; E-Bioscience</td&gt;<td&gt; 00-6993-50</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Anti-mouse CD140a-PE (PDGFRA)</td&gt;<td&gt; E-Bioscience</td&gt;<td&gt; 12-1401-81</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Anti-mouse FITC F4/80-</td&gt;<td&gt; Cederlane</td&gt;<td&gt; CL8940F</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; DMEM w / o Rosso fenolo</td&gt;<td&gt; Gibco</td&gt;<td&gt; 31053</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Collagene di tipo I</td&gt;<td&gt; BD Biosciences</td&gt;<td&gt; 354236</td&gt;<td</td&gt;</tr&gt;</tbody&gt;</table

isolation of normal and cancer-associated fibroblasts from fresh tissues by fluorescence activated cell sorting (facs)

Cancro-associate fibroblasti (CAF) sono il tipo cellulare più prominente nello stroma del tumore di molti tumori, in particolare carcinoma mammario, e la loro presenza prominente è spesso associato con 1,2 prognosi. CAF sono una sottopopolazione di fibroblasti attivati ​​stromali, molti dei quali esprimono il marcatore myofibroblast α-SMA 3. CAF provengono da fibroblasti tissutali locali nonché da midollo osseo, cellule reclutati nel tumore sviluppare e adottare un fenotipo CAF sotto l&#39;influenza del microambiente tumorale 4. CAF sono stati dimostrato di facilitare l&#39;iniziazione del tumore, la crescita e la progressione attraverso la segnalazione che promuove la proliferazione delle cellule tumorali, l&#39;angiogenesi e l&#39;invasione 5-8. Abbiamo dimostrato che i CAF migliorare la crescita tumorale di mediazione che promuove l&#39;infiammazione del tumore, a partire al più presto pre-neoplastiche stadi 9. Nonostante la crescente evidenza dei CAF ruolo chiave svolgere nel facilitare la crescita tumorale,CAF studiano è una continua sfida a causa della mancanza di CAF-specifici marcatori e l&#39;eterogeneità parte di queste cellule, con molti sottotipi coesistenti nel microambiente tumorale 10. Inoltre, studiando fibroblasti in vitro è ostacolato dal fatto che il loro profilo di espressione genica è spesso alterato in coltura tissutale 11,12. Per risolvere questo problema e per consentire imparziale profilo di espressione genica di fibroblasti di topo fresco e tessuti umani, abbiamo sviluppato un metodo basato sui protocolli precedenti per fluorescenza selezione delle cellule attivate (FACS) 13,14. Il nostro approccio si basa su utilizzando PDGFRα come marcatore di superficie per isolare i fibroblasti di topo freschi e tessuti umani. PDGFRα è abbondantemente espresso da entrambi i fibroblasti normali e CAF 9,15. Questo metodo permette di isolare popolazioni pure di fibroblasti normali e CAF, tra cui, ma non limitato a α-SMA + attivato miofibroblasti. Fibroblasti isolati possono quindi essereutilizzati per la caratterizzazione e confronto dell&#39;evoluzione di espressione genica che si verifica nei CAFS durante la tumorigenesi. In effetti, noi e gli altri segnalati profilo di espressione di fibroblasti isolati da cellule ordinamento 16. Questo protocollo è stato eseguito con successo per isolare e creare un profilo popolazioni altamente arricchito di fibroblasti dai tessuti della pelle, della mammella, del pancreas e del polmone. Inoltre, il nostro metodo permette anche di coltura delle cellule filtrate, per eseguire esperimenti funzionali e per evitare contaminazione da cellule tumorali, che spesso è un grosso ostacolo quando si cerca di CAF cultura.

Utilizzando PDGFRα come marker risultati fibroblasti in isolamento delle popolazioni altamente arricchito di fibroblasti tissutali. Il livello di purezza dopo la cernita era del 99%, come quantificato mediante analisi post-liste (Figura 2A). Valutare la percentuale di contaminanti non fibroblasti cellule mediante l&#39;espressione relativa di cellula-specifici geni di controllo (Figura 2B) mostra tipicamente 0,1-0,6% contaminazione. Questo livello di purezza permette di alta profilazio...

Watch this Scientific Journal Video about Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting FACS at JoVE.com

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

Cancro fibroblasti associati (CAF) facilitare l&#39;iniziazione del tumore, la crescita e la progressione attraverso la segnalazione che promuove la proliferazione, l&#39;angiogenesi, e l&#39;infiammazione. Qui si descrive un metodo per isolare popolazioni pure di fibroblasti normali e CAF dal topo freschi e tessuti umani mediante selezione delle cellule, utilizzando PDGFRα come marcatore di superficie.

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 ...

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Biology

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

35.5K Views.  Tel Aviv University. Cancro fibroblasti associati (CAF) facilitare l'iniziazione del tumore, la crescita e la progressione attraverso la segnalazione che promuove la proliferazione, l'angiogenesi, e l'infiammazione. Qui si descrive un metodo per isolare popolazioni pure di fibroblasti normali e CAF dal topo freschi e tessuti umani mediante selezione delle cellule, utilizzando PDGFRα come marcatore di superficie.

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

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite morphogenesis at the functional and molecular levels. To aid in these analyses, we have developed a strategy for the isolation of a subclass of PNS neurons called dendritic arborization (da) neurons that have been widely used for studying dendrite morphogenesis1,2. These neurons are very difficult to isolate as a pure population, due in part to their extremely low occurrence and their difficult-to-reach location below the tough chitinous larval cuticle. Our newly developed method overcomes these challenges, and is based on a fast and specific cell enrichment using antibody-coated magnetic beads. For our magnetic bead sorting studies, we have used age-matched third instar larvae expressing a mouse CD8 tagged GFP fusion protein (UAS-mCD8-GFP)3 under the control of either the class IV dendritic arborization (da) neuron-specific pickpocket (ppk)-GAL4 driver4 or the control of the pan-da neuron-specific GAL421-7 driver5. Although this protocol has been optimized for isolating PNS cells which are attached to the inner wall of the larval cuticle, by varying a few parameters, the same protocol could be used to isolate many different cell types attached to the cuticle at larval or pupal stages of development (e.g. epithelia, muscle, oenocytes etc.), or other cell types from larval organs depending upon the GAL4-specific driver expression pattern. The RNA isolated by this method is of high quality and can be readily used for downstream genomic analyses such as microarray gene expression profiling studies. This approach offers a powerful new tool to perform studies on isolated Drosophila dendritic arborization (da) neurons thereby providing novel insights into the molecular mechanisms underlying dendrite morphogenesis.

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

In this video-article we present a method for the isolation and purification of Drosophila peripheral neurons using a fast magnetic bead assisted cell sorting strategy. RNA obtained from the isolated cells can be readily used for downstream applications including microarray analyses.

Isolamento e purificazione di Drosophila Neuroni periferici da Magnetic Ordinamento Bead

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite ...

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 ...

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

Microbes are major agents mediating the degradation of numerous dissolved organic carbon (DOC) substrates in aquatic environments. However, identification of bacterial taxa that transform specific pools of DOC in nature poses a technical challenge. 
Here we describe an approach that couples bromodeoxyuridine (BrdU) incorporation, fluorescence activated cell sorting (FACS), and 16S rRNA gene-based molecular analysis that allows culture-independent identification of bacterioplankton capable of degrading a specific DOC compound in aquatic environments. Triplicate bacterioplankton microcosms are set up to receive both BrdU and a model DOC compound (DOC amendments), or only BrdU (no-addition control). BrdU substitutes the positions of thymidine in newly synthesized bacterial DNA and BrdU-labeled DNA can be readily immunodetected 1,2. Through a 24-hr incubation, bacterioplankton that are able to use the added DOC compound are expected to be selectively activated, and therefore have higher levels of BrdU incorporation (HI cells) than non-responsive cells in the DOC amendments and cells in no-addition controls (low BrdU incorporation cells, LI cells). After fluorescence immunodetection, HI cells are distinguished and physically separated from the LI cells by fluorescence activated cell sorting (FACS) 3. Sorted DOC-responsive cells (HI cells) are extracted for DNA and taxonomically identified through subsequent 16S rRNA gene-based analyses including PCR, clone library construction and sequencing.

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

Environmental bacterioplankton are incubated with a model dissolved organic carbon (DOC) compound and a DNA labeling reagent, bromodeoxyuridine (BrdU). Afterward, DOC-degrading cells are separated from the bulk community based on their elevated BrdU incorporation using fluorescence activated cell sorting (FACS). These cells are then identified by subsequent molecular analyses.

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 ...

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

Liver fibrosis is defined by the excessive deposition of extracellular matrix by activated myofibroblasts. There are multiple precursors of hepatic myofibroblasts, including hepatic stellate cells, portal fibroblasts and bone marrow derived fibroblasts 1. Hepatic stellate cells have been the best studied, but portal fibroblasts are increasingly recognized as important contributors to the myofibroblast pool, particularly in biliary fibrosis 2. Portal fibroblasts undergo proliferation in response to biliary epithelial injury, potentially playing a key role in the early stages of biliary scarring 3-5. A method of isolating portal fibroblasts would allow in vitro study of this cell population and lead to greater understanding of the role portal fibroblasts play in biliary fibrosis.
Portal fibroblasts have been isolated using various techniques including outgrowth 6, 7 and liver perfusion with enzymatic digestion followed by size selection 8. The advantage of the digestion and size selection technique compared to the outgrowth technique is that cells can be studied without the necessity of passage in culture. Here, we describe a modified version of the original technique described by Kruglov and Dranoff 8 for isolation of portal fibroblasts from rat liver that results in a relatively pure population of primary cells.

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

A technique for isolating portal fibroblasts from rat liver is described. Livers are perfused and digested in situ with collagenase, followed by ex vivo digestion of the liver slurry and size selection of cells. This method provides a pure population of portal fibroblasts without the need for passage in culture.

Isolamento dei fibroblasti Portal ratto<em&gt; In situ</em&gt; Fegato perfusione

Liver fibrosis is defined  by the excessive deposition of extracellular matrix by activated  myofibroblasts. There are multiple precursors of hepatic ...

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 ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

Analisi delle sospensioni cellulari isolate da tessuti solidi da Spectral citometria a flusso

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

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 ...

Fluorescence-Activated Cell Sorting for the Isolation of Scleractinian Cell Populations

Coral reefs are under threat due to anthropogenic stressors. The biological response of coral to these stressors may occur at a cellular level, but the mechanisms are not well understood. To investigate coral response to stressors, we need tools for analyzing cellular responses. In particular, we need tools that facilitate the application of functional assays to better understand how cell populations are reacting to stress. In the current study, we use fluorescence-activated cell sorting (FACS) to isolate and separate different cell populations in stony corals. This protocol includes: (1) the separation of coral tissues from the skeleton, (2) creation of a single cell suspension, (3) labeling the coral cells using various markers for flow cytometry, and (4) gating and cell sorting strategies. This method will enable researchers to work on corals at the cellular level for analysis, functional assays, and gene expression studies of different cell populations.

Corals create biodiverse ecosystems important for both humans and marine organisms. However, we still do not understand the full potential and function of many coral cells. Here, we present a protocol developed for the isolation, labeling, and separation of stony coral cell populations.

Ordinamento cellulare attivato dalla fluorescenza per l'isolamento delle popolazioni cellulari Scleractiniane

Coral reefs are under threat due to anthropogenic stressors. The biological response of coral to these stressors may occur at a cellular level, but the ...

Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the isolation of these vesicles, including ultracentrifugation, ultrafiltration, and size exclusion chromatography. However, not all are suitable for large scale exosome purification and characterization. Outlined here is a protocol for establishing cultures of primary fibroblasts isolated from adult mouse skeletal muscles, followed by purification and characterization of exosomes from the culture media of these cells. The method is based on the use of sequential centrifugation steps followed by sucrose density gradients. Purity of the exosomal preparations is then validated by western blot analyses using a battery of canonical markers (i.e., Alix, CD9, and CD81). The protocol describes how to isolate and concentrate bioactive exosomes for electron microscopy, mass spectrometry, and uptake experiments for functional studies. It can easily be scaled up or down and adapted for exosome isolation from different cell types, tissues, and biological fluids.

This protocol illustrates the 1) the isolation and culture of primary fibroblasts from the adult mouse gastrocnemius muscle as well as 2) purification and characterization of exosomes using a differential ultracentrifugation method combined with sucrose density gradients followed by western blot analyses.

Isolamento e caratterizzazione di esosomi da fibroblasti muscolari scheletrici

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the ...