Questo lavoro è stato finanziato dalla famiglia Smith Award per l&#39;eccellenza nella ricerca biomedica e NIH RO1 AI083426-01 (FW). Patrick Maschmeyer e Melanie Flach sono supportati da borse di dottorato da Boehringer Ingelheim Fondazione.

Topi C57BL / 6 dovrebbero essere usati a ~ 20 settimane di età o più. L&#39;utilizzo di topi maschi del peso 25-30g è raccomandato. La resa di cellule stellate può essere aumentata di alimentazione topi un A-arricchito di vitamina dieta per 2 mesi prima di isolamento delle cellule stellate. Circa 2x10 5 cellule stellate epatiche può essere purificato dal fegato di un C57BL / 6 del mouse, mentre la resa di cellule stellate da topi BALB / c è nettamente superiore. Il protocollo che segue è regolata a 5 topi. La cura degli animali e la sperimentazione sono state eseguite in conformità approvato Animal Care istituzionali e protocolli Comitato Usa. 1. Perfusione in situ di fegato mouse con enzimi digestivi <ol><li> Scaldare il tampone SC1 e soluzioni per perfusioni enzima in un bagno d&#39;acqua a 37 ° C. </li><li> Montare un set di infusione alato sul tubo di silicone di una pompa peristaltica. </li><li> Calibrare la pompa con PBS per ottenere un flusso laminare di 6.5ml/min, che è la portata che saranno utilizzati per la perfusione in situ del fegato. Equilibrare il tubo in silicone con SC1 soluzione. </li><li> Anestetizzare il topo mediante iniezione intraperitoneale di ketamina (90 mg / kg) e xilazina (10 mg / kg) e la soluzione di prova per la perdita di riflessi per garantire la narcotizzazione profondo. </li><li> Fissare il mouse in posizione supina su una base adatta. </li><li> Usare le forbici e pinze per eseguire una incisione longitudinale nella pelle addominale ed esporre il peritoneo. </li><li> Aprire con cautela il peritoneo e spostare l&#39;intestino dalla cavità addominale al lato sinistro dell&#39;animale al fine di esporre la vena porta. </li><li> Inserire la cannula del set per infusione nella vena porta. Per questo passaggio, l&#39;uso di uno stereomicroscopio è raccomandato. </li><li> Avviare la perfusione del fegato con la soluzione SC1 30ml. Aprire la vena cava inferiore subito dopo l&#39;inizio del flusso. Lavaggio di successo del fegato è indicato da una perdita di colore del tessuto del fegato. </li><li> Perfusione del fegato con una soluzione di 30ml E pronasi. Al momento la digestione di successo, i lobi del fegato si gonfierà, i lobuli e appariranno distinte attraverso la capsula. </li><li> Perfusione del fegato con una soluzione di 30ml P Collagenasi. Il fegato a questo punto ha perso la sua forma e aspetto atonica e amorfo. </li><li> Accuratamente separato il fegato dal diaframma e gli organi circostanti e riporlo in 70ml SC2 soluzione sul ghiaccio. </li><li> Ripetere la procedura per topi aggiuntivi. </li></ol> 2. Nella digestione in vitro di fegato del mouse Tutte le fasi ulteriori di lavoro deve essere effettuata in condizioni sterili in una cappa a flusso laminare. <ol><li> Tagliare il fegato a pezzi di circa 2x2x2mm 3 usando forbici affilate. </li><li> Combina la sospensione di 70ml SC2 compresi i pezzi di fegato con 50 ml di pronasi E-Collagenasi soluzione P e aggiungere 1 ml di soluzione di DNasi I. </li><li> Digerire il fegato per 20 minuti a 37 ° C sotto agitazione. </li></ol> 3. Gradiente di densità centrifugazione <ol><li> Filtrare la sospensione cellulare attraverso filtri cellula 70μm in 6 tubi Falcon da 50 ml e aggiungere fino a 50 ml con SC2 buffer. Centrifugare per 10 minuti a 600 grammi e 4 ° C. </li><li> Con attenzione aspirare 40 ml del surnatante, quindi aggiungere 150μl DNasi soluzione che ad ogni provetta e sospendere nuovamente le cellule. </li><li> Si riuniscono gli sospensioni cellulari in 4 tubi Falcon da 50 ml e lavare con GBSS-B, centrifugare per 10 minuti a 600g e 4 ° C. </li><li> Con attenzione aspirare la maggior quantità di surnatante possibile senza disturbare il pellet e aggiungere 150μl DNasi soluzione che ad ogni provetta. Risospendere il pellet cellulare in 10 ml GBSS-B per provetta. </li><li> Piscina le cellule in 2 tubi Falcon da 50 ml e aggiungere GBSS-B per un volume totale di 36ml per provetta. Aggiungere 14 ml soluzione Nycodenz a ciascuna provetta e mescolare bene. </li><li> Trasferire 10 ml di sospensione cellulare in un unico tubo 12ml gradiente di centrifugazione, con conseguente 10 tubi in totale. Delicatamente sovrapposizione la sospensione cellulare con 1,5 ml GBSS-B per provetta. </li><li> Centrifugare i gradienti per 15 minuti a 1500g e 4 ° C senza freno. Successivamente, epatociti verranno pellet sul fondo della provetta mentre le cellule stellate si trovano nella interfase come un anello bianco. </li><li> Accuratamente raccolto l&#39;interfase contenente le cellule stellate e lavarli con GBSS-B; centrifugare per 10 minuti a 600 grammi e 4 ° C. </li><li> Aspirare il surnatante e risospendere le cellule in 20 ml di DMEM supplementato con 10% di siero FBS, 1% HEPES penicillina / streptomicina, 2mM L-Glutammina, piruvato di sodio 1mM, e 10mm. Trasferire le cellule in fiasche coltura tissutale con una concentrazione di 2x10 4 cellule / cm 2. Incubare le cellule a 37 ° C e 5% di CO 2. </li><li> Cambiare il supporto, non appena le cellule stellate epatiche sono aderenti (circa 2 ore dopo la preparazione) per lavare via le cellule morte e detriti cellulari. </li><li> Il giorno seguente, la cellula stellates dovrebbe sviluppare la loro caratteristica forma di stella con morfologia perinucleare vitamina A-la memorizzazione vescicole lipidiche. </li><li> Per gli esperimenti successivi, le cellule stellate epatiche può essere facilmente staccato partire da lastre di superfici in plastica con lievi soluzioni enzimatiche come Accutase, ad esempio, </li></ol> 4. Rappresentante dei risultati: Dopo la preparazione delle cellule stellate epatiche utilizzando il protocollo previsto, la purezza delle popolazioni isolate possono essere testati in considerazione tre caratteristiche principali di questo tipo di cellule, come le stelle, come forma, gocce lipidiche perinucleare, e l&#39;espressione della proteina acidica fibrillare gliale (GFAP ). Immagini rappresentative per l&#39;aspetto caratteristico delle cellule stellate epatiche 2 ore dopo l&#39;isolamento delle cellule, così come il giorno 1 e 3 della coltura in vitro sono rappresentati in figura 1. La Figura 2 mostra una colorazione rappresentante immunofluorescenza per GFAP nelle cellule stellate epatiche, che sono state coltivate per 3 giorni dopo l&#39;isolamento delle cellule. Cellule stellate epatiche si differenziano in cellule myofibroblastic durante la coltura in vitro. Un tratto distintivo caratteristico di queste cellule stellate attivate è l&#39;espressione di actina alfa del muscolo liscio (αSMA). La figura 3 mostra immunofluorescenza per ASMA e miosina IIA in cellule stellate attivate il giorno 7 di coltura in vitro. <img src="/files/ftp_upload/2710/2710fig1.jpg" alt="Figura 1" /> Figura 1. Morfologia caratteristica delle cellule stellate epatiche. Cellule stellate sono state isolate da fegato del mouse utilizzando il protocollo previsto. Le immagini ritraggono le cellule stellate 2 ore dopo l&#39;isolamento delle cellule (a, b), così come il giorno 1 (c) e il giorno 3 (d) della coltura in vitro. Cellule stellate epatiche presentano elevate quantità di vescicole lipidiche nei siti perinucleare e acquistano il loro caratteristico astrale-come morfologia durante i primi giorni di coltura in vitro (ingrandimento 200x). <img src="/files/ftp_upload/2710/2710fig2.jpg" alt="Figura 2" /> Figura 2. Cellule stellate epatiche specificamente esprimere GFAP nel fegato. Cellule stellate sono state isolate e immunofluorescently colorate per GFAP (rosso) il giorno 3 di coltura in vitro. Nuclei delle cellule sono rappresentate in blu (Hoechst macchia). <img src="/files/ftp_upload/2710/2710fig3.jpg" alt="Figura 3" /> Figura 3. Cellule stellate epatiche differenziarsi in miofibroblasti. Cellule stellate epatiche isolate da topi C57BL / 6 sono state coltivate per 7 giorni. Successivamente, sono stati trasferiti alle diapositive da camera e colorate per ASMA (mostrato in rosso) e miosina IIA (rappresentato in verde). I nuclei delle cellule erano di contrasto con Hoechst (blu). <table border="1"><tbody><tr><td colspan="2"> SC1 Buffer </td></tr><tr><td> EGTA </td><td> 190mg </td></tr><tr><td> Glucosio </td><td> 900 mg </td></tr><tr><td> HEPES </td><td> 10 ml di soluzione madre 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> Rosso fenolo </td><td> 6mg </td></tr><tr><td> dH 2 O </td><td> riempire fino a 1l </td></tr><tr><td colspan="2"> SC2 Buffer </td></tr><tr><td> CaCl 2 x 2H 2 O </td><td> 560mg </td></tr><tr><td> HEPES </td><td> 10 ml di soluzione 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> Rosso fenolo </td><td> 6mg </td></tr><tr><td> dH 2 O </td><td> riempire fino a 1l </td></tr><tr><td colspan="2"> GBSS-A Buffer </td></tr><tr><td> KCl </td><td> 370mg </td></tr><tr><td> CaCl 2 x 2H 2 O </td><td> 225mg </td></tr><tr><td> Glucosio </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> Rosso fenolo </td><td> 6mg </td></tr><tr><td> dH 2 O </td><td> riempire fino a 1l </td></tr><tr><td colspan="2">GBSS-B Buffer </td></tr><tr><td> CaCl 2 x 2H 2 O </td><td> 225mg </td></tr><tr><td> Glucosio </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> Rosso fenolo </td><td> 6mg </td></tr><tr><td> dH 2 O </td><td> riempire fino a 1l </td></tr><tr><td colspan="2"> Pronasi Soluzione E perfusione </td></tr><tr><td> E pronasi </td><td> 100mg (4000 PU / mg min) </td></tr><tr><td> SC2 Buffer </td><td> 200ml </td></tr><tr><td colspan="2"> Collagenasi P perfusione Solution </td></tr><tr><td> Collagenasi P </td><td> 85mg (1,78 U / mg lyo) </td></tr><tr><td> SC2 Buffer </td><td> 200ml </td></tr><tr><td colspan="2"> Pronasi E-Solution Collagenasi P </td></tr><tr><td> E pronasi </td><td> 50mg (4000 PU / mg min) </td></tr><tr><td> Collagenasi P </td><td> 85mg (1,78 U / mg lyo) </td></tr><tr><td> SC2 Buffer </td><td> 50ml </td></tr><tr><td colspan="2"> DNasi I Solution </td></tr><tr><td> DNasi I </td><td> 6mg (ca. 2000U/mg) </td></tr><tr><td> GBSS-B </td><td> 3ml </td></tr><tr><td colspan="2"> Nycodenz Soluzione </td></tr><tr><td> Nycodenz </td><td> 8g </td></tr><tr><td> GBSS-A </td><td> 28ml </td></tr></tbody></table> Tabella 1. Tamponi e soluzioni enzima necessario per l&#39;isolamento delle cellule stellate epatiche. Il pH di tutti i buffer dovrebbe essere regolata per 7,3-7,4. Inoltre, filtrazione sterile di tutti i buffer è raccomandato. E &#39;importante adeguare la quantità di enzima utilizzato a seconda delle determinata attività enzimatica.

<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>

Nessun conflitto di interessi dichiarati.

Cellule stellate epatiche regolano essenziali processi fisiologici e fisiopatologici nel fegato. Inoltre, le cellule stellate possiedono proprietà presentanti l&#39;antigene, rendendoli una componente importante della risposta immunitaria epatica. Sebbene le cellule stellate epatiche costituiscono il 10-15% del numero totale di cellule nel fegato, l&#39;isolamento di queste cellule è impegnativo a causa della loro localizzazione nello spazio di Disse perisinusoidal. Qui vi presentiamo un m...

<table border="1"><tbody><tr><th> Nome di reagente </th><th> Azienda </th><th> Catalog No. </th></tr><tr><td> 70μm cella Filtro </td><td> BD </td><td> 352350 </td></tr><tr><td> CaCl 2 x 2H 2 O </td><td> Sigma-Aldrich </td><td> C3306 </td></tr><tr><td> Collagenasi P </td><td> Roche </td><td> 11249002001 </td></tr><tr><td> DMEM </td><td> Gibco </td><td> 11960 </td></tr><tr><td> DNasi I </td><td> Roche </td><td> 10104159001 </td></tr><tr><td> DPBS </td><td> Cellgro </td><td> 21-031-cv </td></tr><tr><td> EGTA </td><td> Fluka </td><td> 3777 </td></tr><tr><td> Siero bovino fetale </td><td> Gibco </td><td> 16000 </td></tr><tr><td> Glucosio </td><td> Sigma-Aldrich </td><td> G7528 </td></tr><tr><td> Gradiente di centrifugazione Tubi </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-Glutammina </td><td> Gibco </td><td> 25030 </td></tr><tr><td> MgCl 2 x 6H 2 O </td><td> Fluka </td><td> 63068 </td></tr><tr><td> MgSO 4 x 7H 2 O </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> Penicillina / streptomicina </td><td> Gibco </td><td> 15140 </td></tr><tr><td> Pompa peristaltica </td><td> Cole Parmer </td><td> HV-7523-70 </td></tr><tr><td> Rosso fenolo </td><td> Sigma-Aldrich </td><td> P4633 </td></tr><tr><td> E pronasi </td><td> Calbiochem </td><td> 7433-2 </td></tr><tr><td> Tubo di silicone </td><td> Masterflex </td><td> HV-96440-14 </td></tr><tr><td> Piruvato di sodio </td><td> Gibco </td><td> 11360 </td></tr><tr><td> Tessuto fiasco cultura (25cm 2) </td><td> BD </td><td> 353108 </td></tr><tr><td> Infusione Set alato </td><td> Terumo </td><td> 1SV27EL </td></tr></tbody></table>

seven steps to stellate cells

Cellule stellate epatiche sono fegato residenti cellule simili a stelle morfologia e si trovano nello spazio di Disse tra fegato e cellule endoteliali sinusoidali 1,2 epatociti. Cellule stellate sono derivati ​​dai precursori del midollo osseo e memorizzare fino a 80% del totale del corpo di vitamina A 1, 2. Dopo l&#39;attivazione, le cellule stellate differenziarsi in miofibroblasti per produrre la matrice extracellulare, contribuendo così alla fibrosi epatica 3. Sulla base delle loro capacità di contrarsi, le cellule stellate myofibroblastic in grado di regolare il tono vascolare associata a ipertensione portale 4. Recentemente, abbiamo dimostrato che le cellule stellate epatiche sono cellule che presentano l&#39;antigene potenti e possono attivare le cellule NKT così come convenzionali linfociti T 5. Qui vi presentiamo un metodo efficiente per la preparazione delle cellule stellate epatiche dal fegato di topo. Grazie alla loro localizzazione perisinusoidal, l&#39;isolamento delle cellule stellate epatiche è un processo multi-step. Al fine di rendere accessibile a cellule stellate isolamento dallo spazio di Disse, fegati mouse sono perfusi in situ con l&#39;pronasi E enzimi digestivi e Collagenasi perfusione P. In seguito, il tessuto epatico viene sottoposto ad ulteriore trattamento enzimatico con pronasi Collagenasi E e P vitro. Successivamente, il metodo sfrutta la massiccia quantità di vitamina A-la memorizzazione gocce lipidiche nelle cellule stellate epatiche. Questa funzione consente la separazione delle cellule stellate da altri tipi di cellule epatiche per centrifugazione su una pendenza dell&#39;8% Nycodenz. Il protocollo qui descritto produce una popolazione altamente pura e omogenea delle cellule stellate. Purezza delle preparazioni può essere valutata dalla colorazione per il marcatore molecola di proteina gliale fibrillare acida (GFAP), prima dell&#39;analisi al microscopio a fluorescenza e citometria a flusso. Inoltre, microscopia luce rivela l&#39;apparizione unica di cellule epatiche stellate a forma di stella che ospitano grandi quantità di goccioline lipidiche. Nel loro insieme, vi presentiamo un protocollo dettagliato per l&#39;isolamento efficiente delle cellule stellate epatiche, comprese le immagini rappresentative del loro aspetto morfologico e di espressione GFAP che contribuiscono a definire l&#39;entità delle cellule stellate.

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

Seven Steps to Stellate Cells

Qui si descrive un metodo per l&#39;isolamento delle cellule stellate epatiche dal fegato di topo. Per la purificazione delle cellule stellate, fegati mouse sono digeriti<em&gt; In situ</em&gt; E<em&gt; In vitro</em&gt; Da pronasi-collagenasi trattamento prima centrifugazione a gradiente di densità. Questa tecnica produce elevata purezza cellule stellate epatiche.

Sette passi per Stellate Cellule

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

Research

JoVE Journal

Immunology and Infection

33.5K Views.  Harvard Medical School. Qui si descrive un metodo per l'isolamento delle cellule stellate epatiche dal fegato di topo. Per la purificazione delle cellule stellate, fegati mouse sono digeriti In situ E In vitro Da pronasi-collagenasi trattamento prima centrifugazione a gradiente di densità. Questa tecnica produce elevata purezza cellule stellate epatiche.

Video: 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.

Valutazione della risposta stomatica a Live cellule batteriche utilizzando Imaging Whole Leaf

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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Immunologia e infezioni

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.

A seguito di Cell-destino<em&gt; E. coli</em&gt; Dopo l&#39;infezione da fago 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.

Un metodo modificato EPA 1623 che utilizza flusso tangenziale a fibre cave di ultrafiltrazione e di calore passi dissociazione per rilevare Waterborne<em&gt; Cryptosporidium</em&gt; E<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.

Trasduzione Adenoviral di CD4 T naive Cells allo Studio Differenziazione 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).

Sviluppo di un modello colite Antigen-driven per studiare presentazione di antigeni da cellule presentanti l&#39;antigene alle cellule 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.

Taglio di precisione polmone mouse Fette per la visualizzazione in diretta cellule dendritiche polmonare

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.

Un metodo integrato per l&#39;analisi della chimotassi rapida dei neutrofili direttamente da una goccia di sangue

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.

Sovrapressione e purificazione dell&#39;uomo<em&gt; Cis</em&gt; -preniltransferasi in<emEscherichia 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 nuovo sistema privo di alimentatore per la produzione di massa di cellule di assassino naturali murini 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.

Sviluppo e validazione di una singola molecola ultrasensibile di matrice digitale analisi enzima-collegata dell'immunosorbente per umana dell'interferone-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 ...