Ce travail a été financé par le Prix de la famille Smith pour l&#39;excellence en recherche biomédicale et les NIH RO1 AI083426-01 (FW). Patrick Maschmeyer et Melanie Flach sont soutenus par des bourses de doctorat de la Fondation de Boehringer Ingelheim.

Souris C57BL / 6 doit être utilisé à ~ 20 semaines d&#39;âge ou plus âgés. L&#39;utilisation de souris mâles pesant 25-30g est recommandée. Le rendement des cellules étoilées peut être augmentée par l&#39;alimentation des souris de la vitamine A régime enrichi pour 2 mois avant l&#39;isolement de cellules étoilées. Environ 2x10 5 cellules étoilées du foie peut être purifié à partir du foie d&#39;un C57BL / 6 de la souris, alors que le rendement des cellules étoilées du souris Balb / c est considérablement plus élevé. Le protocole suivant est ajusté à 5 souris. Et soins des animaux d&#39;expérimentation ont été effectuées en conformité avec soin des animaux et des protocoles approuvés institutionnels Comité utilisation. 1. En perfusion in situ du foie de souris avec des enzymes digestives <ol><li> Chauffer le tampon SC1 et solutions pour perfusions d&#39;enzymes dans un bain d&#39;eau à 37 ° C. </li><li> Monter une perfusion ailée mis sur le tube de silicone d&#39;une pompe péristaltique. </li><li> Calibrer la pompe avec du PBS pour obtenir un écoulement laminaire de 6.5ml/min, qui est le débit qui sera utilisé pour la perfusion in situ dans le foie. Equilibrer le tube en silicone avec SC1 solution. </li><li> Anesthésier la souris par injection intrapéritonéale de kétamine (90 mg / kg) et de xylazine (10 mg / kg) de solution et de test pour la perte des réflexes pour assurer narcotisation profonde. </li><li> Fixer la souris en position couchée sur un support adapté. </li><li> Utilisez des ciseaux et des pinces à effectuer une incision longitudinale dans la peau de l&#39;abdomen et d&#39;exposer le péritoine. </li><li> Ouvrez soigneusement le péritoine et déplacer les intestins hors de la cavité abdominale sur le côté gauche de l&#39;animal afin d&#39;exposer la veine porte. </li><li> Insérer la canule de l&#39;ensemble de perfusion dans la veine porte. Pour cette étape, l&#39;utilisation d&#39;un stéréomicroscope est recommandée. </li><li> Démarrer la perfusion du foie avec la solution SC1 30ml. Ouvrez la veine cave inférieure immédiatement après l&#39;ouverture de la circulation. Succès de rinçage du foie est indiquée par une perte de couleur du tissu hépatique. </li><li> Perfuser le foie avec la solution E Pronase 30ml. Lors de la digestion réussie, les lobes du foie se gonflent, et les lobules apparaissent distincts à travers la capsule. </li><li> Perfuser le foie avec une solution collagénase P 30ml. Le foie à ce point a perdu sa forme et regarde atone et amorphe. </li><li> Soigneusement séparés du foie au diaphragme et les organes environnants et le stocker dans 70ml SC2 solution sur la glace. </li><li> Répétez la procédure pour les souris supplémentaires. </li></ol> 2. Dans la digestion in vitro de foies de souris Toutes les autres étapes de travail devrait être effectué dans des conditions stériles dans une hotte à flux laminaire. <ol><li> Couper les foies en morceaux d&#39;environ 3 2x2x2mm l&#39;aide de ciseaux pointus. </li><li> Combinez la suspension de 70ml SC2 y compris les morceaux de foie avec 50 ml de pronase E-collagénase solution P et ajouter 1 ml de solution de DNase I. </li><li> Recueil des foies pendant 20min à 37 ° C tout en remuant. </li></ol> 3. Centrifugation en gradient de densité <ol><li> Filtrer la suspension des cellules à travers les tamis cellulaire 70μm dans des tubes Falcon 50 ml et 6 ajouter jusqu&#39;à 50ml avec SC2 tampon. Centrifuger pendant 10 minutes à 600g à 4 ° C. </li><li> Aspirer délicatement 40ml de surnageant, puis ajouter 150μl de DNase I solution à chaque tube et remettre les cellules. </li><li> Piscine des suspensions cellulaires en tubes 50ml Falcon 4 et lavez avec GBSS-B, centrifugeuse pour 10min à 600 g et 4 ° C. </li><li> Aspirer délicatement autant que possible le surnageant sans perturber le culot et ajouter 150μl de DNase I solution à chaque tube. Resuspendre les culots cellulaires dans 10 ml GBSS-B par tube. </li><li> Piscine des cellules dans des tubes Falcon 50 ml et ajouter 2 GBSS-B pour un volume total de 36ml par tube. Ajouter 14ml Nycodenz solution à chaque tube et bien mélanger. </li><li> Transfert de 10ml de la suspension de cellules dans un tube de centrifugation en gradient de 12ml, résultant en 10 tubes au total. Doucement superposition de la suspension cellulaire avec 1.5ml GBSS-B par tube. </li><li> Centrifuger les gradients pour 15min à 1500g et 4 ° C sans frein. Par la suite, les hépatocytes sera granulés au fond du tube tandis que les cellules étoilées sont trouvés dans l&#39;interphase comme un anneau blanc. </li><li> Soigneusement la récolte de l&#39;interphase contenant les cellules étoilées et les laver avec GBSS-B; centrifugeuse pendant 10 minutes à 600g à 4 ° C. </li><li> Aspirer le surnageant et remettre en suspension les cellules dans 20 ml de DMEM complété avec inactivé à la chaleur 10% de FBS, HEPES 1% de pénicilline / streptomycine, 2 mM L-glutamine, 1 mM de pyruvate de sodium, et 10mm. Transférer les cellules dans des flacons de culture de tissu avec une concentration de 2x10 4 cellules / cm 2. Incuber les cellules à 37 ° C et 5% de CO 2. </li><li> Changer les médias dès que les cellules étoilées du foie sont adhérentes (environ 2h après la préparation) pour laver les cellules mortes et les débris cellulaires. </li><li> Le jour suivant, la cellule stellaires devraient développer leur caractéristique en forme d&#39;étoile avec la morphologie des vésicules périnucléaires vitamine lipide A-stockage. </li><li> Pour les expériences ultérieures, les cellules étoilées du foie peut être facilement détaché de surfaces en plastique non revêtus en utilisant des solutions douces enzymatiques tels que Accutase, par exemple </li></ol> 4. Les résultats représentatifs: Suite à la préparation des cellules étoilées du foie en utilisant le protocole fourni, la pureté de la population isolée peut être testé considérer trois caractéristiques majeures de ce type cellulaire, comme ressemblant à une étoile forme, gouttelettes lipidiques périnucléaire, et l&#39;expression de protéine acide fibrillaire gliale (GFAP ). Images représentant pour l&#39;aspect caractéristique des cellules étoilées hépatiques 2h après l&#39;isolement cellulaire, ainsi que le jour 1 et 3 de la culture in vitro sont représentés dans la figure 1. La figure 2 montre une coloration représentative d&#39;immunofluorescence pour la GFAP dans les cellules étoilées du foie, qui ont été cultivées pendant 3 jours suivant l&#39;isolement cellulaire. Cellules étoilées du foie se différencier en cellules myofibroblastique au cours de culture in vitro. Une caractéristique caractéristique de ces cellules étoilées activées est l&#39;expression de l&#39;alpha actine musculaire lisse (αSMA). La figure 3 montre immunofluorescence pour la ZGSA et la myosine IIA dans les cellules étoilées activées au jour 7 de la culture in vitro. <img src="/files/ftp_upload/2710/2710fig1.jpg" alt="Figure 1" /> Figure 1. Morphologie caractéristique des cellules étoilées du foie. Cellules étoilées ont été isolés à partir de foies de souris en utilisant le protocole fourni. Les images dépeignent étoilées 2h cellules après l&#39;isolement cellulaire (a, b), ainsi que le jour 1 (c) et le jour 3 (d) de la culture in vitro. Cellules étoilées hépatiques présentent des quantités élevées de vésicules lipidiques sur les sites périnucléaire et acquièrent leur distinctifs astral, comme la morphologie pendant les premiers jours de culture in vitro (grossissement 200x). <img src="/files/ftp_upload/2710/2710fig2.jpg" alt="Figure 2" /> Figure 2 cellules. Étoilées du foie expriment spécifiquement la GFAP dans le foie. Cellules étoilées ont été isolés et immunofluorescently colorées pour la GFAP (en rouge) au jour 3 de culture in vitro. Noyaux cellulaires sont représentées en bleu (Hoechst tache). <img src="/files/ftp_upload/2710/2710fig3.jpg" alt="Figure 3" /> Figure 3 cellules. Étoilées du foie différencient en myofibroblastes. Cellules étoilées hépatiques isolées de souris C57BL / 6 ont été cultivés pendant 7 jours. Par la suite, ils ont été transférés aux diapositives de chambre et colorées pour Asma (en rouge) et la myosine IIA (représentée en vert). Les noyaux des cellules ont été contre avec Hoechst (bleu). <table border="1"><tbody><tr><td colspan="2"> SC1 Tampon </td></tr><tr><td> EGTA </td><td> 190mg </td></tr><tr><td> Le glucose </td><td> 900mg </td></tr><tr><td> HEPES </td><td> 10 ml de solution stock 1M </td></tr><tr><td> KCl </td><td> 400mg </td></tr><tr><td> Na 2 HPO 4 x 2 H 2 O </td><td> 151mg </td></tr><tr><td> NaCl </td><td> 8g </td></tr><tr><td> NaH 2 PO 4 x H 2 O </td><td> 78mg </td></tr><tr><td> NaHCO 3 </td><td> 350mg </td></tr><tr><td> Rouge de phénol </td><td> 6mg </td></tr><tr><td> dH 2 O </td><td> remplir jusqu&#39;à 1l </td></tr><tr><td colspan="2"> SC2 Tampon </td></tr><tr><td> CaCl 2 x 2 H 2 O </td><td> 560mg </td></tr><tr><td> HEPES </td><td> 10ml de solution stock 1M </td></tr><tr><td> KCl </td><td> 400mg </td></tr><tr><td> Na 2 HPO 4 x 2 H 2 O </td><td> 151mg </td></tr><tr><td> NaCl </td><td> 8g </td></tr><tr><td> NaH 2 PO 4 x H 2 O </td><td> 78mg </td></tr><tr><td> NaHCO 3 </td><td> 350mg </td></tr><tr><td> Rouge de phénol </td><td> 6mg </td></tr><tr><td> dH 2 O </td><td> remplir jusqu&#39;à 1l </td></tr><tr><td colspan="2"> GBSS-A Tampon </td></tr><tr><td> KCl </td><td> 370mg </td></tr><tr><td> CaCl 2 x 2 H 2 O </td><td> 225mg </td></tr><tr><td> Le glucose </td><td> 991mg </td></tr><tr><td> KH 2 PO 4 </td><td> 30mg </td></tr><tr><td> MgCl 2 x 6 H 2 O </td><td> 210mg </td></tr><tr><td> MgSO 4 x 7 H 2 O </td><td> 70mg </td></tr><tr><td> Na 2 HPO 4 x 2 H 2 O </td><td> 75mg </td></tr><tr><td> NaHCO 3 </td><td> 227mg </td></tr><tr><td> Rouge de phénol </td><td> 6mg </td></tr><tr><td> dH 2 O </td><td> remplir jusqu&#39;à 1l </td></tr><tr><td colspan="2">GBSS-tampon B </td></tr><tr><td> CaCl 2 x 2 H 2 O </td><td> 225mg </td></tr><tr><td> Le glucose </td><td> 991mg </td></tr><tr><td> KCl </td><td> 370mg </td></tr><tr><td> KH 2 PO 4 </td><td> 30mg </td></tr><tr><td> MgCl 2 x 6 H 2 O </td><td> 210mg </td></tr><tr><td> MgSO 4 x 7 H 2 O </td><td> 70mg </td></tr><tr><td> Na 2 HPO 4 x 2 H 2 O </td><td> 75mg </td></tr><tr><td> NaCl </td><td> 8g </td></tr><tr><td> NaHCO 3 </td><td> 227mg </td></tr><tr><td> Rouge de phénol </td><td> 6mg </td></tr><tr><td> dH 2 O </td><td> remplir jusqu&#39;à 1l </td></tr><tr><td colspan="2"> Solution pronase E perfusion </td></tr><tr><td> Pronase E </td><td> 100mg (4000 PU / mg min) </td></tr><tr><td> SC2 Tampon </td><td> 200ml </td></tr><tr><td colspan="2"> Solution de collagénase perfusion P </td></tr><tr><td> Collagénase P </td><td> 85 mg (1,78 U / mg lyo) </td></tr><tr><td> SC2 Tampon </td><td> 200ml </td></tr><tr><td colspan="2"> Pronase E-collagénase solution p </td></tr><tr><td> Pronase E </td><td> 50mg (4000 PU / mg min) </td></tr><tr><td> Collagénase P </td><td> 85 mg (1,78 U / mg lyo) </td></tr><tr><td> SC2 Tampon </td><td> 50ml </td></tr><tr><td colspan="2"> DNase I Solution </td></tr><tr><td> DNase I </td><td> 6mg (ca. 2000U/mg) </td></tr><tr><td> GBSS-B </td><td> 3ml </td></tr><tr><td colspan="2"> Solution Nycodenz </td></tr><tr><td> Nycodenz </td><td> 8g </td></tr><tr><td> GBSS-A </td><td> 28ml </td></tr></tbody></table> Tableau 1. Tampons et solutions enzyme nécessaire pour l&#39;isolement de cellules étoilées du foie. Le pH de tous les tampons doit être ajustée à 7.3 à 7.4. Par ailleurs, la filtration stérile de tous les tampons est recommandée. Il est important d&#39;adapter la quantité d&#39;enzyme utilisée en fonction de l&#39;activité enzymatique donnée.

<ol>
	<li>Friedman, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+stellate+cells:+protean,+multifunctional,+and+enigmatic+cells+of+the+liver.">Hepatic stellate cells: protean, multifunctional, and enigmatic cells of the liver.</a> Physiol Rev. 88, 125-172 (2008).</li><li>Geerts, A. H. i. s. t. o. r. y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=heterogeneity,+developmental+biology,+and+functions+of+quiescent+hepatic+stellate+cells.">heterogeneity, developmental biology, and functions of quiescent hepatic stellate cells.</a> Semin Liver Dis. 21, 311-335 (2001).</li><li>Gressner, A. M. &. a. m. p. ;. a. m. p., Weiskirchen, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modern+pathogenetic+concepts+of+liver+fibrosis+suggest+stellate+cells+and+TGF-beta+as+major+players+and+therapeutic+targets.">Modern pathogenetic concepts of liver fibrosis suggest stellate cells and TGF-beta as major players and therapeutic targets.</a> J Cell Mol Med. 10, 76-99 (2006).</li><li>Reynaert, H., Thompson, M. G., Thomas, T., Geerts, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+stellate+cells:+role+in+microcirculation+and+pathophysiology+of+portal+hypertension.">Hepatic stellate cells: role in microcirculation and pathophysiology of portal hypertension.</a> Gut. 50, 571-581 (2002).</li><li>Winau, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ito+cells+are+liver-resident+antigen-presenting+cells+for+activating+T+cell+responses.">Ito cells are liver-resident antigen-presenting cells for activating T cell responses.</a> Immunity. 26, 117-129 (2007).</li><li>Minicis, S. D. e. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+profiles+during+hepatic+stellate+cell+activation+in+culture+and+in+vivo.">Gene expression profiles during hepatic stellate cell activation in culture and in vivo.</a> Gastroenterology. 132, 1937-1946 (2007).</li><li>Knittel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+liver+myofibroblasts+and+hepatic+stellate+cells+in+normal+and+diseased+rat+livers:+distinct+roles+of+(myo-)fibroblast+subpopulations+in+hepatic+tissue+repair.">Localization of liver myofibroblasts and hepatic stellate cells in normal and diseased rat livers: distinct roles of (myo-)fibroblast subpopulations in hepatic tissue repair.</a> Histochem Cell Biol. 112, 387-401 (1999).</li></ol>

Cellules étoilées du foie réguler essentielles des processus physiologiques et physiopathologiques dans le foie. Par ailleurs, les cellules étoilées possèdent des propriétés présentant un antigène, les rendant une composante importante de la réponse immunitaire hépatique. Bien que les cellules étoilées hépatiques comprennent 10-15% du nombre total de cellules dans le foie, l&#39;isolement de ces cellules est difficile en raison de leur localisation dans l&#39;espace de Disse périsinusoïdales. <p cla...

<table border="1"><tbody><tr><th> Nom de réactif </th><th> Société </th><th> Catalogue No. </th></tr><tr><td> Passoire cellulaire 70μm </td><td> BD </td><td> 352350 </td></tr><tr><td> CaCl 2 x 2 H 2 O </td><td> Sigma-Aldrich </td><td> C3306 </td></tr><tr><td> Collagénase P </td><td> Roche </td><td> 11249002001 </td></tr><tr><td> DMEM </td><td> Gibco </td><td> 11960 </td></tr><tr><td> DNase I </td><td> Roche </td><td> 10104159001 </td></tr><tr><td> DPBS </td><td> Cellgro </td><td> 21 à 031 cv- </td></tr><tr><td> EGTA </td><td> Fluka </td><td> 3777 </td></tr><tr><td> Sérum de veau fœtal </td><td> Gibco </td><td> 16000 </td></tr><tr><td> Le glucose </td><td> Sigma-Aldrich </td><td> G7528 </td></tr><tr><td> Tubes centrifugation en gradient </td><td> Greiner Bio-One </td><td> 163160 </td></tr><tr><td> HEPES </td><td> Gibco </td><td> 15630 </td></tr><tr><td> KCL </td><td> Sigma-Aldrich </td><td> P9541 </td></tr><tr><td> KH 2 PO 4 </td><td> Sigma-Aldrich </td><td> P9791 </td></tr><tr><td> L-Glutamine </td><td> Gibco </td><td> 25030 </td></tr><tr><td> MgCl 2 x 6 H 2 O </td><td> Fluka </td><td> 63068 </td></tr><tr><td> MgSO 4 7H 2 O x </td><td> Sigma-Aldrich </td><td> M2773 </td></tr><tr><td> Na 2 HPO 4 x 2H 2 O </td><td> Fluka </td><td> 71643 </td></tr><tr><td> NaCl </td><td> Sigma-Aldrich </td><td> S3014 </td></tr><tr><td> NaH 2 PO 4 x H 2 O </td><td> Fluka </td><td> 71507 </td></tr><tr><td> NaHCO 3 </td><td> Fluka </td><td> 71628 </td></tr><tr><td> Nycodenz </td><td> Accudenz AG </td><td> AN7050/BLK </td></tr><tr><td> Pénicilline / streptomycine </td><td> Gibco </td><td> 15140 </td></tr><tr><td> Pompe péristaltique </td><td> Cole Parmer </td><td> HV-7523-70 </td></tr><tr><td> Rouge de phénol </td><td> Sigma-Aldrich </td><td> P4633 </td></tr><tr><td> Pronase E </td><td> Calbiochem </td><td> 7433-2 </td></tr><tr><td> Tube de silicone </td><td> Masterflex </td><td> HV-96440-14 </td></tr><tr><td> Pyruvate de sodium </td><td> Gibco </td><td> 11360 </td></tr><tr><td> Flacon de culture tissulaire (25cm 2) </td><td> BD </td><td> 353108 </td></tr><tr><td> Ensemble de perfusion Winged </td><td> Terumo </td><td> 1SV27EL </td></tr></tbody></table>

seven steps to stellate cells

Cellules étoilées du foie sont le foie-résident des cellules de morphologie en forme d&#39;étoile et sont situés dans l&#39;espace de Disse entre les cellules endothéliales sinusoïdales du foie et de 1,2 hépatocytes. Cellules étoilées sont dérivées à partir de précurseurs de moelle osseuse et de stocker jusqu&#39;à 80% de la vitamine A totale du corps 1, 2. Lors de l&#39;activation des cellules étoilées se différencier en myofibroblastes pour produire la matrice extracellulaire, contribuant ainsi à 3 fibrose hépatique. Basé sur leur capacité à se contracter, les cellules étoilées myofibroblastique peut réguler le tonus vasculaire associée à l&#39;hypertension portale 4. Récemment, nous avons démontré que les cellules étoilées du foie sont des cellules présentant l&#39;antigène puissants et peuvent activer les cellules NKT ainsi que les lymphocytes T conventionnels 5. Nous présentons ici une méthode pour la préparation efficace des cellules étoilées du foie à partir du foie de souris. En raison de leur localisation périsinusoïdales, l&#39;isolement de cellules étoilées du foie est un processus multi-étapes. Afin de rendre accessibles les cellules étoilées à l&#39;isolement de l&#39;espace de Disse, foies de souris sont perfusées in situ avec le E enzymes digestives pronase et la collagénase P. perfusion Après, le tissu hépatique est soumis à un traitement enzymatique supplémentaire avec pronase E et P dans la collagénase vitro. Par la suite, la méthode tire parti de l&#39;énorme quantité de gouttelettes lipidiques de vitamine A-stockage dans les cellules étoilées du foie. Cette fonctionnalité permet la séparation des cellules étoilées du foie d&#39;autres types de cellules par centrifugation sur un gradient de 8% Nycodenz. Le protocole décrit ici donne une population très pure et homogène de cellules étoilées. Pureté de la préparation peut être évaluée par coloration de la molécule marqueur protéique fibrillaire gliale acide (GFAP), avant l&#39;analyse par microscopie à fluorescence ou cytométrie en flux. En outre, la microscopie optique révèle l&#39;aspect unique de la forme d&#39;étoile cellules étoilées du foie qui abritent de grandes quantités de gouttelettes lipidiques. Pris ensemble, nous présentons un protocole détaillé pour l&#39;isolement efficace des cellules étoilées du foie, y compris des images représentatives de leur aspect morphologique et d&#39;expression GFAP qui aident à définir l&#39;entité de cellules étoilées.

Watch this Scientific Journal Video about Seven Steps to Stellate Cells at JoVE.com

Seven Steps to Stellate Cells

Nous décrivons ici une méthode pour l&#39;isolement de cellules étoilées du foie à partir du foie de souris. Pour la purification de cellules étoilées, les foies de souris sont digérés<em&gt; In situ</em&gt; Et<em&gt; In vitro</em&gt; Par la pronase-collagénase traitement préalable à la centrifugation en gradient de densité. Cette technique rendements très pure cellules étoilées du foie.

Sept étapes pour Stellate Cellules

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

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Immunology and Infection

33.5K vues.  Harvard Medical School. Nous décrivons ici une méthode pour l'isolement de cellules étoilées du foie à partir du foie de souris. Pour la purification de cellules étoilées, les foies de souris sont digérés In situ Et In vitro Par la pronase-collagénase traitement préalable à la centrifugation en gradient de densité. Cette technique rendements très pure cellules étoilées du foie.

Vidéo: Seven Steps to Stellate Cells

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of guard cells, which are able to close the stomatal pore in response to a number of external factors including light intensity, carbon dioxide concentration, and relative humidity (RH). The stomatal pore is also the main route for pathogen entry into leaves, a crucial step for disease development. Recent studies have unveiled that closure of the pore is effective in minimizing bacterial disease development in Arabidopsis plants; an integral part of plant innate immunity. Previously, we have used epidermal peels to assess stomatal response to live bacteria (Melotto et al. 2006); however maintaining favorable environmental conditions for both plant epidermal peels and bacterial cells has been challenging. Leaf epidermis can be kept alive and healthy with MES buffer (10 mM KCl, 25 mM MES-KOH, pH 6.15) for electrophysiological experiments of guard cells. However, this buffer is not appropriate for obtaining bacterial suspension. On the other hand, bacterial cells can be kept alive in water which is not proper to maintain epidermal peels for long period of times. When an epidermal peel floats on water, the cells in the peel that are exposed to air dry within 4 hours limiting the timing to conduct the experiment. An ideal method for assessing the effect of a particular stimulus on guard cells should present minimal interference to stomatal physiology and to the natural environment of the plant as much as possible. We, therefore, developed a new method to assess stomatal response to live bacteria in which leaf wounding and manipulation is greatly minimized aiming to provide an easily reproducible and reliable stomatal assay. The protocol is based on staining of intact leaf with propidium iodide (PI), incubation of staining leaf with bacterial suspension, and observation of leaves under laser scanning confocal microscope. Finally, this method allows for the observation of the same live leaf sample over extended periods of time using conditions that closely mimic the natural conditions under which plants are attacked by pathogens.

We have developed a simple and reproducible protocol to access stomatal response to live bacteria. This method minimizes wounding and manipulation of the leaf as compared to the use of epidermal peels reported previously.

Évaluer la réponse stomatique à Live cellules bactériennes en utilisant l&#39;imagerie feuilles entières

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of ...

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Immunologie et infection

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

Après Cell-sort<em&gt; E. coli</em&gt; Après infection par le phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

A Modified EPA Method 1623 that Uses Tangential Flow Hollow-fiber Ultrafiltration and Heat Dissociation Steps to Detect Waterborne Cryptosporidium and Giardia spp.

Cryptosporidium and Giardia species are two of the most prevalent protozoa that cause waterborne diarrheal disease outbreaks worldwide. To better characterize the prevalence of these pathogens, EPA Method 1623 was developed and used to monitor levels of these organisms in US drinking water supplies 12. The method has three main parts; the first is the sample concentration in which at least 10 L of raw surface water is filtered. The organisms and trapped debris are then eluted from the filter and centrifuged to further concentrate the sample. The second part of the method uses an immunomagnetic separation procedure where the concentrated water sample is applied to immunomagnetic beads that specifically bind to the Cryptosporidium oocysts and Giardia cysts allowing for specific removal of the parasites from the concentrated debris. These (oo)cysts are then detached from the magnetic beads by an acid dissociation procedure. The final part of the method is the immunofluorescence staining and enumeration where (oo)cysts are applied to a slide, stained, and enumerated by microscopy.
Method 1623 has four listed sample concentration systems to capture Cryptosporidium oocysts and Giardia cysts in water: Envirochek filters (Pall Corporation, Ann Arbor, MI), Envirochek HV filters (Pall Corporation), Filta-Max filters (IDEXX, Westbrook, MA), or Continuous Flow Centrifugation (Haemonetics, Braintree, MA). However, Cryptosporidium and Giardia (oo)cyst recoveries have varied greatly depending on the source water matrix and filters used1,14. A new tangential flow hollow-fiber ultrafiltration (HFUF) system has recently been shown to be more efficient and more robust at recovering Cryptosporidium oocysts and Giardia cysts from various water matrices; moreover, it is less expensive than other capsule filter options and can concentrate multiple pathogens simultaneously1-3,5-8,10,11. In addition, previous studies by Hill and colleagues demonstrated that the HFUF significantly improved Cryptosporidium oocysts recoveries when directly compared with the Envirochek HV filters4. Additional modifications to the current methods have also been reported to improve method performance. Replacing the acid dissociation procedure with heat dissociation was shown to be more effective at separating Cryptosporidium from the magnetic beads in some matrices9,13 .
This protocol describes a modified Method 1623 that uses the new HFUF filtration system with the heat dissociation step. The use of HFUF with this modified Method is a less expensive alternative to current EPA Method 1623 filtration options and provides more flexibility by allowing the concentration of multiple organisms.

A Modified EPA Method 1623 that Uses Tangential Flow Hollow-fiber Ultrafiltration and Heat Dissociation Steps to Detect Waterborne Cryptosporidium and Giardia spp.

This protocol describes the use of a tangential flow hollow-fiber ultrafiltration sample concentration system and a heat dissociation as alternative steps for the detection of waterborne Cryptosporidium and Giardia species using EPA Method 1623.

Une méthode mise à jour EPA 1623 qui utilise tangentielle débit à fibres creuses d&#39;ultrafiltration et de chaleur étapes de dissociation pour la détection de voie d&#39;eau<em&gt; Cryptosporidium</em&gt; Et<em&gt; Giardia spp.</em

Cryptosporidium and Giardia species are two of the most prevalent protozoa that cause waterborne diarrheal disease outbreaks worldwide. To better ...

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 T cells in vitro with TCR- and co-stimulation in the presence of TGF&beta; and IL-2. This bears enormous potential for future therapies, however, the molecules and signaling pathways that control differentiation are largely unknown.
Primary T cells can be manipulated through ectopic gene expression, but common methods fail to target the most important naive state of the T cell prior to primary antigen recognition. Here, we provide a protocol to express ectopic genes in naive CD4 T cells in vitro before inducing Treg differentiation. It applies transduction with the replication-deficient adenovirus and explains its generation and production. The adenovirus can take up large inserts (up to 7 kb) and can be equipped with promoters to achieve high and transient overexpression in T cells. It effectively transduces naive mouse T cells if they express a transgenic Coxsackie adenovirus receptor (CAR). Importantly, after infection the T cells remain naive (CD44low, CD62Lhigh) and resting (CD25-, CD69-) and can be activated and differentiated into Tregs similar to non-infected cells. Thus, this method enables manipulation of CD4 T cell differentiation from its very beginning. It ensures that ectopic gene expression is already in place when early signaling events of the initial TCR stimulation induces cellular changes that eventually lead into Treg differentiation.

Adenoviral gene transfer into naive CD4 T cells with transgenic expression of the Coxsackie adenovirus receptor enables the molecular analysis of regulatory T cell differentiation in vitro.

Transduction Adenoviral de CD4 naïfs cellules T pour étudier la différenciation Treg

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 ...

Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological mechanisms involved during the disease is the T cell transfer model of colitis. In this model, immunodeficient mice (RAG-/- recipients) are reconstituted with naive CD4+ T cells from healthy wild type hosts.
This model allows examination of the earliest immunological events leading to disease and chronic inflammation, when the gut inflammation perpetuates but does not depend on a defined antigen. To study the potential role of antigen presenting cells (APCs) in the disease process, it is helpful to have an antigen-driven disease model, in which a defined commensal-derived antigen leads to colitis. An antigen driven-colitis model has hence been developed. In this model OT-II CD4+ T cells, that can recognize only specific epitopes in the OVA protein, are transferred into RAG-/- hosts challenged with CFP-OVA-expressing E. coli. This model allows the examination of interactions between APCs and T cells in the lamina propria.

In this antigen-driven colitis model, OT-II CD4+ T cells expressing a red fluorescent protein were adoptively transferred into RAG-/- mice that express a green fluorescent protein in mononuclear phagocytes (MPs). The hosts were challenged with Escherichia coli (E.coli) expressing the ovalbumin protein (OVA) fused to a cyan fluorescent protein (CFP).

Développement d&#39;un modèle Colite antigène conduit à étudier la présentation des antigènes par les cellules présentatrices d&#39;antigènes aux cellules T

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological ...

Precision-cut Mouse Lung Slices to Visualize Live Pulmonary Dendritic Cells

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for quantitative analysis of cell surface proteins on immune cells, but it provides no information on the localization and migration patterns of these cells within the lung. Similarly,&#160;chemotaxis assays can be performed to study the potential of cells to respond to chemotactic factors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these aforementioned techniques, the location of individual cell types within the lung can be readily visualized by generating Precision-cut Lung Slices (PCLS), staining them with commercially available, fluorescently tagged antibodies, and visualizing the sections by confocal microscopy. PCLS can be used for both live and fixed lung tissue, and the slices can encompass areas as large as a cross section of an entire lobe. We have used this protocol to successfully visualize the location of a wide variety of cell types in the lung, including distinct types of dendritic cells, macrophages, neutrophils, T cells and B cells, as well as structural cells such as lymphatic, endothelial, and epithelial cells. The ability to visualize cellular interactions, such as those between dendritic cells and T cells, in live, three-dimensional lung tissue, can reveal how cells move within the lung and interact with one another at steady state and during inflammation. Thus, when used in combination with other procedures, such as flow cytometry and quantitative PCR, PCLS can contribute to a comprehensive understanding of cellular events that underlie allergic and inflammatory diseases of the lung.

We describe a method for generating Precision-cut Lung Slices (PCLS) and immunostaining them to visualize the localization of various immune cell types in the lung. Our protocol can be extended to visualize the location and function of many different cell types under a variety of conditions.

coupe de précision de la souris Tranches Lung pour visualiser les cellules vivantes pulmonaire dendritiques

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for ...

An All-on-chip Method for Rapid Neutrophil Chemotaxis Analysis Directly from a Drop of Blood

Neutrophil migration and chemotaxis are critical for our body's immune system. Microfluidic devices are increasingly used for investigating neutrophil migration and chemotaxis owing to their advantages in real-time visualization, precise control of chemical concentration gradient generation, and reduced reagent and sample consumption. Recently, a growing effort has been made by the microfluidic researchers toward developing integrated and easily operated microfluidic chemotaxis analysis systems, directly from whole blood. In this direction, the first all-on-chip method was developed for integrating the magnetic negative purification of neutrophils and the chemotaxis assay from small blood volume samples. This new method permits a rapid sample-to-result neutrophil chemotaxis test in 25 min. In this paper, we provide detailed construction, operation and data analysis method for this all-on-chip chemotaxis assay with a discussion on troubleshooting strategies, limitations and future directions. Representative results of the neutrophil chemotaxis assay testing a defined chemoattractant, N-Formyl-Met-Leu-Phe (fMLP), and sputum from a chronic obstructive pulmonary disease (COPD) patient, using this all-on-chip method are shown. This method is applicable to many cell migration-related investigations and clinical applications.

This article provides the detailed method of performing a rapid neutrophil chemotaxis assay by integrating the on-chip neutrophil isolation from whole blood and the chemotaxis test on a single microfluidic chip.

Une méthode intégrale pour l&#39;analyse rapide de la chimiotaxie des neutrophiles directement à partir d&#39;une goutte de sang

Neutrophil migration and chemotaxis are critical for our body's immune system. Microfluidic devices are increasingly used for investigating neutrophil ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

La surexpression et la purification de l&#39;être humain<em&gt; Cis</em&gt; -prenyltransférase dans<em&gt; Escherichia coli</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

Un nouveau système exempt d’alimentation pour la Production de masse des cellules tueuses naturelles murins In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&amp;#945;

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

Développement et Validation d’une seule molécule ultrasensible de tableau numérique Enzyme-linked Immunosorbent Assay for Human interféron-α

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...