Nous remercions le Dr Yitzhak Oschry et le Dr Orit Sagi-Asif pour leur aide avec le tri par FACS. Cette recherche a été financée par des subventions à NE du Programme de l&#39;Union européenne septième programme-cadre (FP7/2007-2013) en vertu de l&#39;accord de subvention n ° [276890], de l&#39;Association israélienne contre le cancer (# 20110078) et du Fonds de recherche sur le cancer Israël (Recherche Prix ​​du développement de carrière).

1. Dissection du tissu mammaire ou de la peau de souris <ol><li> Avant de disséquer le tissu désiré de souris, préparer les fournitures et réactifs suivants: Fournitures: <ul><li> Outils de chirurgie (lavés avec du détergent, puis trempé dans l&#39;éthanol à 70%). </li><li> Surface de styromousse pour reposer la souris sur lors de la dissection. </li><li> Épingles ou des aiguilles de tenir des souris lors de la dissection. </li><li> Bocal en verre avec couvercle pour la digestion, contenant un barreau d&#39;agitation magnétique (autoclave). </li><li> Bain d&#39;eau à 37 ° C, contenant un agitateur magnétique submersible et suffisamment d&#39;eau pour immerger le pot digestion, tout en reposant sur la plaque d&#39;agitation. </li></ul> Réactifs: <ul><li> Tampon FACS I: (4 ° C): phosphate de Dulbecco saline tamponnée CMF (Calcium Magnésium gratuit) + 0,5% de BSA </li><li> Solution de collagénase (faite immédiatement avant utilisation): 0,05 g collagénase II 0,05 g collagénase IV 0,01 g Deoxyribonucbail 20 ml de tampon FACS I. Conserver la solution de collagénase sur la glace jusqu&#39;à utilisation. </li><li> PharmLyse (Réactif de Lyse chlorure d&#39;ammonium). </li><li> Tampon FACS II: phosphate de Dulbecco saline tamponnée CMF + 1% de SVF. </li></ul></li><li> Dissection des glandes mammaires (voir aussi 17). <ul><li> Euthanasier les souris femelles à l&#39;aide d&#39;inhalation de dioxyde de carbone (CO 2). </li><li> Sur une surface de polystyrène, placez la souris sur son dos et la broche fermement les quatre membres avec 25 aiguilles de calibre (figure 1A). Vaporisez généreusement la souris avec de l&#39;éthanol à 70%. </li><li> En utilisant des pinces, tirer vers le haut la peau de l&#39;abdomen sur la ligne médiane et faire une petite incision avec des ciseaux pointus. </li><li> A partir de l&#39;incision, couper la peau jusqu&#39;au cou de l&#39;animal tout en évitant la perforation des cavités abdominale ou thoracique. </li><li> Coupez la peau sur les pattes arrière de l&#39;incision médiane vers la jambe, ce qui entraîne une &quot;forme de Y&quot;. </li><li> Tirez la peau loin de mouse corps et la broche vers le bas, exposant les glandes mammaires rattachées à la face inférieure de la peau (figure 1B). </li><li> Utilisez Q-tips séparer doucement les glandes mammaires de la peau tout en réduisant tissu conjonctif avec de petits ciseaux bien aiguisés pour séparer la glande mammaire vers la colonne vertébrale de la souris. </li><li> Les glandes abdominales (# 4, 5) sont les meilleurs pour ce protocole. Vous pouvez également utiliser la glande mammaire 2 ème, mais gardez à l&#39;esprit que ces glandes sont anatomiquement situé à proximité du muscle pectoral qui peuvent être difficiles à séparer, résultant en des cellules d&#39;origine mixte du tissu. </li><li> Remarque: Lors de la dissection du tissu tumoral, il est recommandé de supprimer des régions nécrotiques. </li></ul></li><li> La dissection des tissus de la peau: La meilleure façon d&#39;obtenir des tissus de la peau, sans avoir à traiter avec l&#39;épilation est d&#39;utiliser oreilles de souris. Si une plus grande quantité de tissu est nécessaire, vous pouvez vous raser les poils du torse de la souris et de disséquer la peau. <ul><li> Euthanasier les souris uchanter inhalation de CO 2. </li><li> Avec des ciseaux pointus, couper les deux oreilles et les placer immédiatement dans un pot de digestion contenant un volume faible (~ 0,5 ml) de PBS sur la glace. </li></ul></li></ol> 2. Préparation de la glande mammaire de la peau / Suspension Single Cell <ul><li> Placez les glandes mammaires / peau dans un bocal contenant la digestion d&#39;un petit volume de PBS sur la glace. Si le tissu est sanglante lavage avec du PBS x2, puis jetez excès de PBS. </li><li> Hacher soigneusement avec des ciseaux courbes. </li><li> Ajouter (note: cette quantité de solution de collagénase se dissocier efficacement environ 5 g de tissu mammaire ou une tumeur et les oreilles d&#39;un maximum de 15 souris) 20 ml solution de collagénase. </li><li> Placez immédiatement sur plaque d&#39;agitation à 37 ° C bain d&#39;eau, et incuber pendant 15 min en remuant au taux moyen. </li></ul> Remarque: Les durées optimales d&#39;incubation peut varier si la digestion d&#39;autres tissus. <ul><li> Pour arrêter la réaction, on ajoute 30 ml de DMEM froid + F 10%CS. </li><li> Filtrer le mélange tissu / support à travers un tamis 70 um cellule placée au-dessus d&#39;un tube conique de 50 ml. </li><li> Centrifuger le tube conique pendant 5 min à 450 xg à 4 ° C. </li></ul> 3. Lyse des globules rouges Remarque: cette étape n&#39;est pas nécessaire si les souris étaient de cœur perfusé avec du PBS avant leur sacrifice. <ul><li> Aspirer le surnageant à l&#39;aide d&#39;une pipette en verre attaché à vide, sans perturber le culot cellulaire. </li><li> Pour lyser RBC, remettre en suspension culot cellulaire dans une solution à 10 PharmLyse ml et incuber pendant 5 min à température ambiante. </li><li> Neutraliser la solution PharmLyse par addition d&#39;environ 40 ml de tampon FACS I. </li><li> Faites tourner le tube conique avec des cellules pendant 5 min à 450 xg à 4 ° C. </li><li> Aspirer le surnageant </li></ul> 4. Blocage de Fc endogène <ul><li> En option: compter les cellules pour déterminer le volume approprié pour la ste suivanteps. </li><li> Resuspendre les cellules dans 1-3 ml de tampon FACS I (selon le nombre de cellules, et la quantité d&#39;anticorps que vous souhaitez utiliser). </li><li> Ajouter FcBlock (anti-souris CD16/CD32) pour atteindre dilution 1:50 (10 pg / ml) et incuber sur de la glace pendant 10-20 &#39;. Pas besoin de rincer FcBlock. </li></ul> 5. Tachant <ul><li> Sortez aliquotes de 100 pi dans des tubes eppendorf être utilisées comme contrôle non teinté et des couleurs simples (selon le nombre d&#39;anticorps fluorescents utilisés). </li><li> Pour fibroblastes étiquettes: ajoutez anti-PDGFRα (directement conjugué à un fluorophore) à une dilution de 1:50 (10 pg / ml). </li><li> Pour marquer d&#39;autres populations cellulaires (par exemple, des cellules immunitaires, les macrophages, les cellules endothéliales, etc): ajouter appropriées anticorps par fluorescence conjugués à des marqueurs de surface, à une dilution de 1:50. </li></ul> Remarque: bien que PDGFRα est un marqueur robuste pour les fibroblastes, il est recommandé d&#39;inclure des anticorps pour immune cellules et de cellules épithéliales afin d&#39;exclure toutes les populations doubles positifs et renforcer ainsi la pureté de fibroblastes isolés. <ul><li> Ajouter 2 l de chaque anticorps de chacun des tubes eppendorf de contrôle de couleur uniques. </li><li> Incuber les cellules avec des anticorps pendant 1 heure sur la glace dans l&#39;obscurité. </li><li> Pour laver: remplir les tubes avec du tampon FACS I et tourner pendant 5 min à 450 xg à 4 ° C. </li><li> Aspirer le surnageant, remettre les cellules en FACS Je tampon à une concentration d&#39;environ 2 x 10 6 cellules / ml, ou n&#39;importe quel concentration la plus appropriée pour le tri avec votre trieur FACS disponibles. </li></ul> En option: Les cellules peuvent être remises en suspension dans du tampon FACS II pour la durée de tri. Cela peut améliorer la viabilité des cellules. Toutefois, l&#39;exposition à des facteurs présents dans le sérum peut avoir un effet l&#39;expression des gènes, qui doivent être testés, et pris en compte. <ul><li> Transfert cellules marquées dans des tubes FACS avec filtre haut: filtre les cellules dans les tubes àempêcher amas cellulaires. </li><li> Pour exclure les cellules mortes, ajouter 7AAD (0,5 pg / ml) ou l&#39;iodure de propidium (PI) à des échantillons (sauf pour le contrôle non teinté). </li></ul> Remarque: Pour éviter de perdre des cellules, vous pouvez ajouter 7AAD/PI à l&#39;échantillon non taché après l&#39;enregistrement dans les FACS, et utiliser à nouveau comme 7AAD seule commande. <ul><li> Conserver les échantillons sur la glace lors de l&#39;analyse. </li></ul> 6. Isolement de cellules par FACS (Remarque: cette partie du protocole nécessite la connaissance de l&#39;exploitation d&#39;un trieur FACS, par exemple BD FACS AriaII, ou l&#39;assistance d&#39;un technicien qualifié). <ul><li> Utilisation du logiciel trieur FACS (BD FACSDiva), préparer des parcelles d&#39;analyse appropriés pour analyser diffusion vers l&#39;avant (FSC), la diffusion latérale (SSC), 7AAD, FITC, PE et tout autre fluorophore utilisé pour marquer les cellules (figure 2). </li><li> Avant de charger chaque échantillon au trieur de cellules, vortex brièvement pour remettre en suspension les cellules. </li><li> Commencez par analytion de l&#39;échantillon sans tache, afin de régler le déclenchement de la population cellulaire totale, à la porte des débris cellulaires, et de déterminer autofluorescence ou coloration de fond. </li><li> Analyser l&#39;échantillon non coloré + 7AAD de déterminer et porte la population de cellules vivantes de la population cellulaire totale. </li><li> Analyser chaque contrôle seule couleur de déterminer et de calibrer les paramètres tels que la rémunération. </li><li> Analyser l&#39;échantillon coloré pour définir la position des portes pour le tri. </li><li> Une fois les paramètres et portails pour les populations de cellules aient été définies, commencer à trier les populations cellulaires marqués dans des tubes Eppendorf ou 15 ml tubes coniques contenant un milieu de culture ou de tampon de lyse ARN (comme RLT tampon ou Trizol). </li></ul> Remarque: Si le tri d&#39;un grand nombre de cellules, il n&#39;est pas recommandé de trier directement dans un tampon de lyse ARN, comme le volume de liquide de gainage qui accompagne les cellules se diluer le tampon de lyse et de réduire le rendement. <ul><li> Spin cellules vers le bas immédiatement après le tri est terminé, et le transfert dans un milieu frais ou tampon de lyse ARN, selon le but de votre expérience. </li><li> Si le tri afin de cultiver des cellules isolées, effectuez sorte stérile. Pour une meilleure récupération des cellules, utilisez le rouge de phénol libre DMEM + 5% SVF au lieu de tampon FACS II et recueillir des cellules dans un milieu de culture avec 20% de FCS. </li><li> Si la culture de fibroblastes triés, alwaysplate sur des plaques recouvertes de collagène et jamais directement sur le plastique car cela conduit à l&#39;activation des fibroblastes entraînant des changements dans leur expression génique. </li></ul>

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Aucun conflit d&#39;intérêt déclaré.

Bien que les expériences réalisées en culture de tissus peut être informatif et proposer des principes fonctionnels qui peuvent être vérifiées in vivo, il est connu que des changements importants se produisent dans l&#39;expression des gènes des cellules en culture 11,12. Afin d&#39;éviter une étape de culture de tissus lors du profilage d&#39;expression génique dans les fibroblastes, nous avons développé un protocole qui permet l&#39;isolement de la normale, ainsi que des fibroblastes a...

<table border="1"&gt;<tbody&gt;<tr&gt;<td<strong&gt; Nom du réactif</strong</td&gt;<td<strong&gt; Société</strong</td&gt;<td<strongNuméro de catalogue&gt;</strong</td&gt;<td<strong&gt; Commentaires (optionnel)</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; Secteurs d&#39;activité biologique</td&gt;<td&gt; 02-023-1A</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Collagénase II</td&gt;<td&gt; Worthington</td&gt;<td&gt; LS4176</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Collagénase IV</td&gt;<td&gt; Worthington</td&gt;<td&gt; LS4188</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Désoxyribonucléase</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;<tdTamis cellulaire&gt; 70 pm</td&gt;<td&gt; SPL</td&gt;<td&gt; 93070</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Purifié anti-souris 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 sonde (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-souris 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-souris 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 rouge de phénol</td&gt;<td&gt; Gibco</td&gt;<td&gt; 31053</td&gt;<td</td&gt;</tr&gt;<tr&gt;<tdCollagène de type&gt; 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)

Fibroblastes associés au cancer (CAF) sont le type cellulaire le plus important au sein du stroma tumoral de nombreux cancers, notamment dans les cancers du sein, et leur présence importante est souvent associée à un mauvais pronostic 1,2. CAF sont une sous-population de fibroblastes du stroma activé, dont beaucoup expriment le marqueur de myofibroblastes α-SMA 3. CAF proviennent de fibroblastes tissulaires locales ainsi que de la moelle osseuse des cellules dérivées recrutés dans la tumeur en développement et adopter un phénotype CAF sous l&#39;influence du microenvironnement tumoral 4. CAF a montré une tumeur afin de faciliter l&#39;initiation, la croissance et la progression à travers de signalisation qui favorise la prolifération cellulaire tumorale, l&#39;angiogenèse et l&#39;invasion 5-8. Nous avons démontré que les CAF améliorer la croissance tumorale par la médiation de l&#39;inflammation tumorale-promotion, en commençant au plus tôt le pré-néoplasiques étapes 9. En dépit des preuves croissantes des CAF rôle clé que jouent dans la facilitation de la croissance tumorale,CAF étudiant a été un défi permanent en raison de l&#39;absence de marqueurs spécifiques à la CAF et de l&#39;hétérogénéité majorité de ces cellules, avec de nombreux sous-types de co-existantes dans le microenvironnement de la tumeur 10. Par ailleurs, l&#39;étude de fibroblastes in vitro est entravée par le fait que leur profil d&#39;expression génique est souvent altérée dans 11,12 culture de tissus. Pour résoudre ce problème et permettre impartiale profil d&#39;expression génique de fibroblastes de souris frais et tissus humains, nous avons développé une méthode basée sur des protocoles antérieurs pour le tri cellulaire activé par fluorescence (FACS) 13,14. Notre approche repose sur l&#39;utilisation de PDGFRα comme un marqueur de surface à isoler les fibroblastes de souris frais et les tissus humains. PDGFRα est abondamment exprimé par les deux fibroblastes normaux et CAF 9,15. Cette méthode permet d&#39;isoler des populations pures de fibroblastes normaux et les EFC, y compris, mais sans s&#39;y limiter α-SMA + activé myofibroblastes. Fibroblastes isolés peuvent ensuite êtreutilisé pour la caractérisation et la comparaison de l&#39;évolution de l&#39;expression des gènes qui se produit en cours de tumorigénèse CAF. En effet, nous et d&#39;autres ont rapporté profilage de l&#39;expression des fibroblastes isolés par tri cellulaire 16. Ce protocole a été réalisé avec succès pour isoler et caractériser les populations hautement enrichies de fibroblastes de la peau des tissus, mammaires, le pancréas et les poumons. De plus, notre méthode permet également de cultiver des cellules triées, afin de réaliser des expériences fonctionnelles et pour éviter toute contamination par des cellules tumorales, ce qui est souvent un obstacle important lorsque vous essayez de CAF culture.

PDGFRα utilisant comme marqueur de résultats de fibroblastes dans l&#39;isolement de populations de fibroblastes hautement enrichi tissus. Le niveau de pureté après le tri a été de 99%, telle que quantifiée par l&#39;analyse post-tri (figure 2A). Estimer le pourcentage de contamination des cellules non fibroblastiques par l&#39;expression relative de gènes spécifiques des cellules de contrôle (figure 2B) montre généralement une contamination 0,1-0,6%. Ce niveau de pureté é...

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

Cancer fibroblastes associés (CAF) de faciliter l&#39;initiation des tumeurs, la croissance et la progression à travers signalisation qui favorise la prolifération, l&#39;angiogenèse et l&#39;inflammation. Nous décrivons ici une méthode pour isoler des populations pures de fibroblastes normaux et de souris CAF frais et tissus humains par tri cellulaire, en utilisant PDGFRα comme un marqueur de surface.

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 ...

isolation-normal-cancer-associated-fibroblasts-from-fresh-tissues

Research

JoVE Journal

Biology

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

35.5K vues.  Tel Aviv University. Cancer fibroblastes associés (CAF) de faciliter l'initiation des tumeurs, la croissance et la progression à travers signalisation qui favorise la prolifération, l'angiogenèse et l'inflammation. Nous décrivons ici une méthode pour isoler des populations pures de fibroblastes normaux et de souris CAF frais et tissus humains par tri cellulaire, en utilisant PDGFRα comme un marqueur de surface.

Vidéo: 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.

Isolement et purification de Drosophile Neurones périphériques en triant bille magnétique

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

Recherche

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 ...

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.

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 ...

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.

Isolement des fibroblastes de rat par portail<em&gt; In situ</emPerfusion du foie&gt;

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.

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 ...

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.

Analyse des suspensions de cellules isolées de tissus solides par Spectral cytométrie en flux

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.

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 ...

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.

Tri cellulaire activée par fluorescence pour l’isolement des populations de cellules sclérosiennes

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.

Isolement et caractérisation d’exosomes à partir de fibroblastes musculaires squelettiques

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