Finanziato in parte dalla concessione NIH RO1AI015503. Topi infettati di schistosome erano forniti dal NIAID schistosomiasi Resource Center presso l'Istituto di ricerca biomedica (Rockville, MD) attraverso NIH-NIAID contratto HHSN272201000005I per la distribuzione attraverso le risorse della BEI.

Tutti cura degli animali e procedure sperimentali sono state approvate dal comitato di uso della University of Wisconsin-Madison sotto protocollo istituzionale Animal Care e non. V005717. Il seguente protocollo comporta lavorare in una struttura di livello 2 di biosicurezza (BSL2) per agenti patogeni umani, anche se nessuna delle fasi di schistosome raffigurate in questo protocollo sono infettiva per l'uomo o altri mammiferi. Seguire politiche istituzionali per la gestione degli agenti patogeni umani del gruppo di rischio 2 (RG2).
Nota: Usiamo topi femmina Swiss-Webster (6 - settimana vecchi) a causa della loro maggiore suscettibilità all'infezione10, ottenendo quindi più grandi oneri di uovo. Tucker et al. descrivere i protocolli di infezione mouse e lumaca utilizzati in questo modello sistema11. La Tabella materiali elenca tutte le attrezzature specifiche, materiali, reagenti e fonti animali necessari per svolgere questo protocollo sperimentale.
1. il trattamento dei fegati infetti
<ol><li>Eutanasia topi, post-infezione 6-7 settimane, da asfissia di CO2 (tasso di 10-30% del volume della camera al minuto). Topi di monitor per la cessazione della respirazione e battito cardiaco. Nota: In genere fino a 20 topi possono essere elaborati in un dato momento.</li>
	<li>Dopo aver trasferito eutanasizzati topi una cappa di biosicurezza, posizionare il mouse sulle loro spalle e spruzzare loro superfici ventrale con etanolo al 70%. Lasciate macerare per 1 min.</li>
	<li>Pizzicare e tirare la pelle dell'addome più basso e tagliare tutta la base della pelle pizzicata più vicina all'addome con forbici chirurgiche sterili. Tirare la pelle tagliata anteriormente (cioè, verso la testa) per rivelare il fegato. Si noti che il fegato dovrebbe essere ampliato e screziato dovuto una risposta granulomatosa indotta da uovo (Figura 2A).</li>
	<li>Rimuovere il fegato e posizionarlo in un becher contenente 200 mL di sterile 1,2% NaCl soluzione contenente pen/strep.</li>
	<li>Ripetere i passaggi da 1.3 e 1.4 con topi restanti. Nota: Venti topi sono generalmente trattati in un solo raccolto.</li>
	<li>Posizionare i fegati in una capsula di Petri sterile e rimuovere non-fegato grasso/connettivo con pinze sterili.</li>
	<li>Trasferimento fegatini tagliati/puliti in una bottiglia sterile 250 mL Centrifuga contenente ~ 200 mL di soluzione sterile di NaCl 1,2% con penna/strep.</li>
	<li>Vigorosamente agitare il flacone contenente i fegati e, utilizzando una pipetta monouso sterile, rimuovere la superficie in eccesso schiuma/detriti galleggianti. Lentamente versare fuori la soluzione salina rimanente in un contenitore o affondare contenenti candeggina 1% (disinfettante).</li>
	<li>Aggiungi ~ 200 mL di soluzione salina fresca per i fegati e ripetere il passaggio 1.8 tre volte di più.</li>
</ol>2. fegato isolamento di estrazione e miracidial
<ol><li>Inserire i fegatini in una tazza piccola sterili in acciaio inox frullatore e aggiungere ~ 2 mL di soluzione di 1,2% di NaCl (Figura 2B).</li>
	<li>Coprire la tazza con un foglio e una capsula di Petri di evitare fuoriuscite e mescolare per 1 min dall'alternarsi di bassa e alta velocità (20 s bassa velocità/10 s ad alta velocità; ripetizione) (Figura 2).</li>
	<li>Distribuire uniformemente la sospensione del fegato blended in bottiglie sterili 250 mL centrifuga (per 20 fegati utilizzano 4 bottiglie).</li>
	<li>Aggiungere soluzione fisiologica sterile con antibiotici a ~ 200 mL bottiglia/volume totale. Pesare/equilibrio bottiglie prima della centrifugazione.</li>
	<li>Centrifugare i fegati mescolati a 290 x g per 15 min a 4 oC. delicatamente versare i surnatanti in un contenitore con candeggina prima dello smaltimento.</li>
	<li>Ripetere i passaggi da 2.4-2.5 una volta. Essere sicuri Risospendere accuratamente sedimento del fegato prima della centrifugazione.</li>
	<li>Dopo aver scartato l'ultima soluzione fisiologica lavare (passo 2,6), aggiungere 200 mL di acqua sterile stagno con penna/strep al tessuto del fegato pellettato, agitare per risospendere il sedimento e trasferire la sospensione in un pallone volumetrico da sterilizzata a 1-L che è stato completamente rivestito di alluminio lamina, fatta eccezione per la parte superiore 3 cm del collo (Figura 2D). Nota: Utilizzare due bottiglie (= 10 fegati) per 1-L fiaschetta. Laghetto d'acqua simula l'ambiente d'acqua dolce naturale necessaria per stimolare miracidial Cova da uova.</li>
	<li>Utilizzando acqua sterile dello stagno, riempire il matraccio a circa 3 cm sopra la lamina che copre il collo. Poi, senza ritardo (come miracidia presto cominciano a covare), rimuovere la schiuma residua e tessuto del fegato galleggiante alla superficie pipettando rapidamente fino ~ 12 mL di acqua di stagno contenente detriti e scartando in un separato scartare il tubo. Sostituire con acqua fresca e sterile dello stagno.</li>
	<li>Ripetere la pulizia passo 2.8, altre tre volte, verificare presenza di miracidia nel tubo di scartare. Cessare la ripetizione della fase di pulizia se miracidia appaiono nella soluzione di lavaggio.</li>
	<li>Dopo l'ultimo lavaggio, aggiungere lentamente ~ 12 mL di acqua calda sterile stagno (pre-riscaldato a 30 oC) per creare un gradiente di temperatura con acqua tiepida pulita e sterile sulla parte superiore. Nota: Ciò avviene meglio scaricando lentamente l'acqua lungo il lato del collo per evitare di miscelare boccetta.</li>
	<li>Posizionare una piccola capsula di Petri copertura sopra l'apertura della boccetta e brillare una luce brillante nella parte superiore del pallone sopra la pellicola di copertura per illuminare la superficie superiore dell'acqua. Nota: In genere, entro 5-10 min, nuoto miracidia, attirata dalla luce, si concentrerà in gran numero entro i primi 2-3 cm di acqua di stagno (Figura 3A).</li>
</ol>3. Miracidial raccolta e coltivazione
<ol><li>Quando miracidia hanno accumulato sulla superficie dopo 10 min, utilizzando una pipetta da trasferimento sterile, rimuovere ~ 8 mL di acqua di stagno e trasferirlo in una provetta sterile 15 mL centrifuga. Collocare la provetta contenente miracidia sul ghiaccio.</li>
	<li>Aggiungere posteriore 8 mL di acqua calda sterile stagno lungo l'interno del matraccio tarato e attendere altri 10 min.</li>
	<li>Utilizzando tubi di nuovi ogni volta, ripetere i passaggi da 3.1 e 3.2 altre tre volte. Dopo la raccolta dith 4, tenere il tubo finale in ghiaccio per 10 minuti consentire la miracidia a stabilirsi. Questo set di tubi comprende il primo raccolto.</li>
	<li>Centrifugare le provette contenenti miracidia a 290 x g per 1 min a 4 oC in una centrifuga refrigerata pre-raffreddata.</li>
	<li>Rimuovere immediatamente la maggior parte del surnatante (~ 7 mL) facendo attenzione a non disturbare il pellet di parassita (Figura 3B).</li>
	<li>Piscina tutti la miracidia da questo primo raccolto in un unico tubo, quindi sciacquare tutti i tubi originali con ~ 1 mL di acqua sterile stagno e aggiungere alla provetta del "pool".</li>
	<li>Consentire i parassiti a stabilirsi sul ghiaccio per ~ 5 min e centrifugare nuovamente i parassiti a 290 x g per 1 min a 4 oC.</li>
	<li>Con attenzione rimuovere il supernatante e aggiungere che 6-8 mL sterile Chernin equilibrata soluzione salina (CBSS +; Vedi tabella materiali) contenente pen/strep.</li>
	<li>Delicatamente sospendere la miracidia e aliquota in una piastra di coltura 24 pozzetti (1 mL per pozzetto), seguita dal tubo di risciacquo in 6-8 mL CBSS + e aggiungendo 1 mL di risciacquo in ciascun pozzetto. Incubare i parassiti a 26 oC in condizioni di normossia. Concentrazioni di miracidia isolato potranno variare, ma generalmente in media ~ 7000/mL.</li>
	<li>Se i parassiti sono ancora concentrato presso la sorgente di luce, ripetere i passaggi 3.1 a 3.9 per continuare la raccolta tardiva-cova miracidia ma aumentare l'intervallo di tempo tra le raccolte per 15-20 min. Nota: Questo nuovo set di tubi rappresenta la seconda raccolta. Tuttavia, poiché i numeri miracidial sono in genere Bassi, larve da tutte le provette di solito sono raggruppate in un'unica cultura ben contenenti 2 mL CBSS +.</li>
	<li>Alle 24 ore post-raccolta e coltivazione, Risospendere delicatamente sporocisti nei loro pozzi di pipettaggio.</li>
	<li>Attendere qualche minuto per consentire i parassiti a depositarsi sul fondo dei pozzetti. Una volta che si sono insediati, rimuovere con cautela il surnatante (~1.5 mL), che conterrà la maggior parte delle piastre epidermiche ciliate capannone da miracidia durante la trasformazione larvale. Questa "trasformazione" supernatante può essere scartata o incorporata in studi di follow-up di prodotti secretivi larvali.</li>
	<li>Aggiungere 1,5 mL di fresco CBSS + in ciascun pozzetto e delicatamente pipetta per risospendere sporocisti. Ripetere il passaggio 3.12 se si desidera ulteriore lavaggio o cambiando in un supporto diverso.</li>
</ol>

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Miracidia di S. mansoni, isolato e manipolato come descritto nel presente documento, può infettare solo l'ospite intermedio di lumaca e pertanto non rappresentano un rischio biologico umano durante questa fase di sviluppo larvale. Tuttavia, per evitare l'accidentale esposizione/infezione di lumache, dovrebbe prestare attenzione per eseguire gli isolamenti miracidial in una posizione diversa dalle zone dove la specie di lumaca Biomphalaria sensibili possono essere presente o mantenuto. Una stanza separa...

L'essere umano sangue flukes Schistosoma spp., sono gli agenti causali della schistosomiasi, una malattia tropicale trascurata che affligge un stimato 230 milioni di persone in tutto il mondo1. La specie più diffusa, Schistosoma mansoni, è geograficamente distribuita in Sud America, l'arcipelago dei Caraibi, Medio Oriente e Africa sub-sahariana2. Il ciclo di vita di S. mansonie altri schistosomes, è complesso, che coinvolge un mammifero ospite definitivo e lumache d'acqua dolce del genere Biomphalaria che fungono da ospiti intermedi obbligati.
S. mansoni adulto maschio e femmina a vite senza fine coppie vivono nelle vene mesenteriche posteriore di riprodurre il mammifero ospite, determinando il rilascio di uova embrionate (ovuli) che sono alloggiati in piccole venule della mucosa intestinale. Uova quindi rotture attraverso le pareti del vaso, migrano attraverso il tessuto mucoso e alla fine inserire il lume intestinale dove essi sono annullate con le feci. Quando gli ovuli digitare tratteggio di larve d'acqua dolce e liberamente natante (miracidia), devono, in breve tempo, trovare una lumaca Biomphalaria adatta per infettare al fine di continuare il suo ciclo di vita. Questo processo di infezione coinvolge miracidial attaccamento alla superficie esterna di lumaca seguita da penetrazione attiva e voce della larva nell'ospite. Presto dopo l'entrata, il miracidium inizia a capannone relative piastre epidermiche ciliati esterni come si forma un sincizio che diventerà la nuova superficie esterna (tegumento) della prossima fase larvale, lo sporociste primario o madre. Attraverso la riproduzione asessuata, ogni sporociste primario produce e rilascia una seconda generazione di sporocisti, definiti secondari o sporocisti di figlia, che a sua volta, generano e rilasciare grandi quantità di intramolluscan fase finale, le cercarie di Fasciolopsis3 . Al momento della fuga dall'host lumaca, liberamente natante cercarie sono capaci di associare a e direttamente penetrando la pelle di un uomo o altri host mammiferi adatto. All'entrata in loro nuovo ospite, cercarie trasformano alla fase schistosomula parassitarie e invadono il sistema vascolare. Essi, a loro volta, migrare al polmone e quindi alla vena epatica dove maturano in vermi adulti, formano coppie maschili e femminile e viaggiare a vene mesenteriche per completare il loro ciclo di vita.
Successo intramolluscan o "fase di lumaca" sviluppo larvale schistosomes è essenziale per la continuazione del ciclo di trasmissione interumana host-to-host. Di fondamentale importanza è la seguente voce periodo del miracidium liberamente natante in host lumaca e la sua trasformazione iniziale alla fase primaria sporociste. A seconda della compatibilità fisiologica e immunologica tra ospite e parassita, è durante questa fase di sviluppo larvale che iniziale successo o il fallimento di stabilire infezioni è determinato4,5,6 . Lo sviluppo successivo di sporocisti primari in grado di produrre asessualmente sporocisti secondari, che quindi generano cercarie umani-infettivo, ulteriormente richiedono un ambiente di host fisiologico permissiva che fornisce per tutta crescita del parassita e riproduttiva ha bisogno.
Fin qui, piccolo è conosciuto circa il sottostante fisiologici, biochimici e processi molecolari controllare interazioni di schistosome-lumaca, soprattutto le molecole e vie coinvolte nella regolazione della riproduzione e sviluppo larvale o modulante risposta immune dell'ospite. Pronto accesso a un gran numero di questi stadi larvali in vitro condizioni di coltura che permettono la manipolazione sperimentale faciliterebbe notevolmente alla scoperta delle vie di sviluppo e meccanismi di fondo della crescita cellulare, differenziazione e riproduzione. Percorsi critici parassita o meccanismi, identificati attraverso la sperimentazione in vitro , quindi potrebbero essere utilizzati per interrompere la crescita/riproduzione larvale nell'host di lumaca, o per identificare meccanismi immuni dell'ospite lumaca coinvolti nel riconoscimento e nell'eliminazione di fasi miracidial o sporociste.
In questo articolo e il video di presentazione, forniamo una descrizione dettagliata di un metodo per isolare un numero elevato di nuotare liberamente S. mansoni miracidia per introduzione nella cultura in vitro che poi può essere sottoposto a follow-up sperimentale manipolazione (vedere la Figura 1 per una descrizione schematica del flusso di lavoro di protocollo). Anche se le procedure simili sono state descritte in precedenza7,8,9, abbiamo sentito che un protocollo dettagliato sarebbe utile per i ricercatori che desiderano impiegare questo modello per affrontare domande ancora senza risposta legate alla lo sviluppo larvale intramolluscan, proliferazione cellulare e interazioni immuni di schistosome-lumaca. Inoltre, questo approccio potrebbe essere facilmente adattato per l'isolamento delle uova da altri trematodi medicamente importanti quali vescica-dimora S. haematobium, distomi del fegato o trematodi del polmone.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Chernin&#39;s balanced salt solution (CBSS+)</td>
			<td></td>
			<td></td>
			<td>For 1L of solution</td>
		</tr>
		<tr>
			<td>2.8 g sodium chloride</td>
			<td>Fisher Scientific</td>
			<td>S271-3</td>
			<td>Dissolve salts, except calcium chloride, and&#160;</td>
		</tr>
		<tr>
			<td>0.15 g of potassium chloride&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>P5405</td>
			<td>sugars in 800 mL ddH2O</td>
		</tr>
		<tr>
			<td>0.07 g sodium phosphate, dibasic anhydrous</td>
			<td>Fisher Scientific</td>
			<td>S374-500</td>
			<td>Dissolve calcium chloride separately in 200&#160;</td>
		</tr>
		<tr>
			<td>0.45 g magnesium sulfate heptahydrate&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>M1880</td>
			<td>mL ddH2O</td>
		</tr>
		<tr>
			<td>0.53 g calcium chloride dihydrate&#160;</td>
			<td>Mallinckrodt</td>
			<td>4160</td>
			<td>Slowly add calcium soln to the salt/sugar soln with&#160;</td>
		</tr>
		<tr>
			<td>0.05 g sodium bicarbonate&#160;&#160;</td>
			<td>Fisher Scientific</td>
			<td>S233-3</td>
			<td>with constant mixing</td>
		</tr>
		<tr>
			<td>1 g glucose</td>
			<td>MP Biomedicals</td>
			<td>152527</td>
			<td>Adjust to pH 7.2 and filter sterilize using a&#160;</td>
		</tr>
		<tr>
			<td>1 g trehalose</td>
			<td>Sigma-Aldrich</td>
			<td>T0167</td>
			<td>0.22 &#181;m disposable bottle-top filter</td>
		</tr>
		<tr>
			<td>10 mL 100X penicillin/streptomycin</td>
			<td>Hyclone</td>
			<td>SV30010</td>
			<td>Add filtered penicillin and streptomycin soln&#160; 
			prior use</td>
		</tr>
		<tr>
			<td>Incomplete Bge&#160; medium (Ibge)</td>
			<td></td>
			<td></td>
			<td>For 900 mL solution</td>
		</tr>
		<tr>
			<td>220 mL Schneider&#8217;s Drosophila medium modified</td>
			<td>Lonza</td>
			<td>04-351Q</td>
			<td>Mix Schneider&#39;s medium with 680 mL ddH2O</td>
		</tr>
		<tr>
			<td>4.5 g lactalbumin enzymatic hydrolysate</td>
			<td>Sigma-Aldrich</td>
			<td>L9010</td>
			<td>Add lactalbumin hydrolysate and galactose</td>
		</tr>
		<tr>
			<td>1.3 g galactose</td>
			<td>Sigma-Aldrich</td>
			<td>G0625</td>
			<td>Adjust to pH 7.2 and filter sterilize using a 0.22 &#181;m 
			pre-sterilized disposable bottle-top filter</td>
		</tr>
		<tr>
			<td>Complete Bge medium (cBge)</td>
			<td></td>
			<td></td>
			<td>For 100 mL of solution</td>
		</tr>
		<tr>
			<td>90 mL Incomplete Bge medium</td>
			<td></td>
			<td></td>
			<td>To heat-inactivate FBS: Incubate thawed FBS in&#160;</td>
		</tr>
		<tr>
			<td>9 mL heat-inact. fetal bovine serum (FBS) (Optima)</td>
			<td>Atlanta Biologicals</td>
			<td>S12450</td>
			<td>waterbath at 60&#176;C for 1 hr while gently&#160;</td>
		</tr>
		<tr>
			<td>1 mL 100X penicillin/streptomycin</td>
			<td>Hyclone</td>
			<td>SV30010</td>
			<td>swirling the bottle every 10 min 
			Aliquot heat-inactivated FBS into 15-mL tubes&#160; 
			and store at -20&#176;C.&#160; Mix medium + FBS and 
			filter sterilize using a 0.22 &#181;m pre-sterilized&#160; 
			disposable bottle-top filter&#160; 
			Add penicillin and streptomycin prior to use</td>
		</tr>
		<tr>
			<td>Pond water (stock solution)</td>
			<td></td>
			<td></td>
			<td>1L of stock solution</td>
		</tr>
		<tr>
			<td>12.5 g calcium carbonate</td>
			<td>Fisher Scientific</td>
			<td>C64-500</td>
			<td>Mix all salts in 1L of ddH2O</td>
		</tr>
		<tr>
			<td>1.25 g magnesium carbonate</td>
			<td>Fisher Scientific</td>
			<td>M27-500</td>
			<td>Note that the salts will not have completely&#160;</td>
		</tr>
		<tr>
			<td>1.25 g&#160; sodium chloride</td>
			<td>Fisher Scientific</td>
			<td>S271-3</td>
			<td>dissolved.&#160; Shake vigorously to suspend&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
		</tr>
		<tr>
			<td>0.25 g&#160; potassium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>P5405</td>
			<td>salts prior to making the working soln&#160;&#160;</td>
		</tr>
		<tr>
			<td>Pond water (working solution)</td>
			<td></td>
			<td></td>
			<td>1.5L of solution</td>
		</tr>
		<tr>
			<td>0.8 mL stock solution pond water (shake prior use) in&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td></td>
			<td></td>
			<td>Mix stock to ddH2O</td>
		</tr>
		<tr>
			<td>1500 mL of ddH2O</td>
			<td></td>
			<td></td>
			<td>Sterilize pond water by autoclaving (slow&#160;cycle)</td>
		</tr>
		<tr>
			<td>1.5 mL of 100X penicillin/streptomycin&#160;</td>
			<td>Hyclone</td>
			<td>SV30010</td>
			<td>Add penicillin and streptomycin prior use</td>
		</tr>
		<tr>
			<td>Saline solution (1.2% NaCl)</td>
			<td></td>
			<td></td>
			<td>1.5L of solution</td>
		</tr>
		<tr>
			<td>18 g sodium chloride in 1500 mL of ddH2O</td>
			<td>Fisher Scientific</td>
			<td>S271-3</td>
			<td>Autoclave saline solution to sterilize</td>
		</tr>
		<tr>
			<td>1.5 mL of 100X penicillin/ streptomycin</td>
			<td>Hyclone</td>
			<td>SV30010</td>
			<td>Add penicillin and streptomycin prior use</td>
		</tr>
		<tr>
			<td>Additional equipment and material:&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>7-L mouse euthanizing chamber</td>
			<td></td>
			<td></td>
			<td>Following approved IACUC protocol no. V001551</td>
		</tr>
		<tr>
			<td>Mice</td>
			<td>Taconic Biosciences</td>
			<td></td>
			<td>Swiss-Webster, female, 6-wk old, murine&#160;&#160;</td>
		</tr>
		<tr>
			<td>CO2 tank and regulator</td>
			<td></td>
			<td></td>
			<td>pathogen-free</td>
		</tr>
		<tr>
			<td>24-well tissue culture plate</td>
			<td>TPP</td>
			<td>92424</td>
			<td></td>
		</tr>
		<tr>
			<td>1-L volumetric flasks</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Light source (150W)</td>
			<td>Chiu Tech Corp</td>
			<td>Model F0-150</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge, refrigerated, swinging bucket</td>
			<td>Eppendorf</td>
			<td>Model 5810R</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge bottles (250 mL)</td>
			<td>Nalgene</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>15-mL centrifuge tubes, sterile</td>
			<td>Corning</td>
			<td>430053</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile disposable transfer pipets&#160;</td>
			<td>Fisher Scientific</td>
			<td>1371120</td>
			<td></td>
		</tr>
		<tr>
			<td>0.22 &#181;m pre-sterilized disposable bottle-top filter&#160;</td>
			<td>EMD Millipore</td>
			<td>SCGPS05RE</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless steel blender</td>
			<td>Waring Commercial</td>
			<td>Model 51BL31</td>
			<td></td>
		</tr>
		<tr>
			<td>Blender cup, 100 mL capacity</td>
			<td>Waring Commercial</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted compound microscope</td>
			<td>Nikon Instruments&#160;</td>
			<td>Eclipse TE300</td>
			<td></td>
		</tr>
	</tbody>
</table>

mass isolation and in vitro cultivation of intramolluscan stages of the human blood fluke schistosoma mansoni

Umani sangue trematodi, Schistosoma spp., hanno un ciclo di vita complesso che coinvolge fasi dello sviluppo sessuale e asessuale all'interno di un lumaca intermedi e di mammifero ospite finale, rispettivamente. La capacità di isolare e sostenere le fasi del ciclo di vita diverso in condizioni di coltura in vitro ha notevolmente facilitato le indagini dei meccanismi cellulari, biochimici e molecolari che regolano l'host, la sviluppo e la crescita del parassita interazioni farmacologiche. Trasmissione della schistosomiasi richiede la riproduzione asessuata e lo sviluppo di più stadi larvali all'interno dell'host lumaca; dal miracidium infettiva, attraverso sporocisti primarie e secondarie, alla fase finale cercarial che è infettivo agli esseri umani. In questa carta presentiamo un protocollo passo-passo per massa schiusa e isolamento di Schistosoma mansoni miracidia da uova ottenute dai fegati di topi infetti e la loro successiva introduzione in coltura in vitro . Si prevede che il protocollo dettagliato incoraggerà nuovi ricercatori per partecipare e ampliare questo importante settore della ricerca di schistosome.

La stragrande maggioranza dei miracidia in genere vi sono state raccolte nei primi due "raccolti". Incubazione di miracidia isolato in CBSS + innesca il processo di trasformazione di miracidium-a-sporociste (Figura 4A- C). Entro la prima ora seguente trasferimento alla cultura pozzetti contenente CBSS +, la maggior parte di cessare miracidia nuoto (Figura 4A). A 6 h nella cultura, miracidia stanno attivamente spa...

Watch this Scientific Journal Video about Mass Isolation and In Vitro Cultivation of Intramolluscan Stages of the Human Blood Fluke Schistosoma Mansoni at JoVE.com

Mass Isolation and In Vitro Cultivation of Intramolluscan Stages of the Human Blood Fluke Schistosoma Mansoni

Questo articolo descrive un protocollo per l'isolamento axenica su larga scala e la raccolta di nuotare liberamente miracidia del trematode umano sangue Schistosoma mansoni e la loro successiva elaborazione per l'introduzione in coltura in vitro .

Massa isolamento e coltivazione In Vitro delle fasi Intramolluscan del trematode umano sangue Schistosoma Mansoni

Human blood flukes, Schistosoma spp., have a complex life cycle that involves asexual and sexual developmental phases within a snail intermediate and ...

mass-isolation-vitro-cultivation-intramolluscan-stages-human-blood

Research

JoVE Journal

Developmental Biology

Mass Isolation and In Vitro Cultivation of Intramolluscan Stages of the Human Blood Fluke Schistosoma Mansoni

9.1K Views.  University of Wisconsin, School of Veterinary Medicine. Questo articolo descrive un protocollo per l'isolamento axenica su larga scala e la raccolta di nuotare liberamente miracidia del trematode umano sangue Schistosoma mansoni e la loro successiva elaborazione per l'introduzione in coltura in vitro .

Video: Mass Isolation and In Vitro Cultivation of Intramolluscan Stages of the Human Blood Fluke Schistosoma Mansoni

Isolation of Human Mesenchymal Stem Cells and their Cultivation on the Porous Bone Matrix

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific biomaterials. This work presents a novel option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) that employs bovine bone matrix Nukbone (NKB) as a scaffold. Thus, the application of MSCs in repair and tissue regeneration processes depends principally on the efficient implementation of the techniques for placing these cells in a host tissue. For this reason, the design of biomaterials and cellular scaffolds has gained importance in recent years because the topographical characteristics of the selected scaffold must ensure adhesion, proliferation and differentiation into the desired cell lineage in the microenvironment of the injured tissue. This option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) employs bovine bone matrix as a cellular scaffold and is an efficient culture technique because the cells respond to the topographic characteristics of the bovine bone matrix Nukbone (NKB), i.e., spreading on the surface, macroporous covering and colonizing the depth of the biomaterial, after the cell isolation process. We present the procedure for isolating and culturing MSCs on a bovine matrix.

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific biomaterials. This work presents a novel option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) that employs the bovine bone matrix Nukbone (NKB) as a scaffold.

Isolamento di cellule staminali mesenchimali umane e la loro coltivazione in poroso Bone Matrix

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific ...

Ricerca

Biologia dello sviluppo

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

Analisi delle larve Zebrafish muscolo scheletrico Integrità con Blu di Evans Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; for example, how the zygote polarizes to establish the body axis and how the embryo specifies various cell fates during organ formation. This manuscript describes an in vitro ovule culture method to perform live-cell imaging of developing zygotes and embryos of Arabidopsis thaliana. The optimized cultivation medium allows zygotes or early embryos to grow into fertile plants. By combining it with a poly(dimethylsiloxane) (PDMS) micropillar array device, the ovule is held in the liquid medium in the same position. This fixation is crucial to observe the same ovule under a microscope for several days from the zygotic division to the late embryo stage. The resulting live-cell imaging can be used to monitor the real-time dynamics of zygote polarization, such as nuclear migration and cytoskeleton rearrangement, and also the cell division timing and cell fate specification during embryo patterning. Furthermore, this ovule cultivation system can be combined with inhibitor treatments to analyze the effects of various factors on embryo development, and with optical manipulations such as laser disruption to examine the role of cell-cell communication.

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

This manuscript describes an in vitro ovule cultivation method that enables live-cell imaging of Arabidopsis zygotes and embryos. This method is utilized to visualize the intracellular dynamics during zygote polarization and the cell fate specification in developing embryos.

In Vitro Coltivazione dell'ovulo per Live cell Imaging di zigote polarizzazione e Patterning di embrione in Arabidopsis thaliana

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; ...

Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and colleagues1. Later, others documented the existence of similar types of EPCs originating from bone marrow2,3. More recently, Yoder and Ingram showed that EPCs derived from umbilical cord blood had a higher proliferative potential compared to ones isolated from adult peripheral blood4,5,6. Apart from being involved in postnatal vasculogenesis, EPCs have also shown promise as a cell source for creating tissue-engineered vascular and heart valve constructs7,8. Various isolation protocols exist, some of which involve the cell sorting of mononuclear cells (MNCs) derived from the sources mentioned earlier with the help of endothelial and hematopoietic markers, or culturing these MNCs with specialized endothelial growth medium, or a combination of these techniques9. Here, we present a protocol for the isolation and culture of EPCs using specialized endothelial medium supplemented with growth factors, without the use of immunosorting, followed by the characterization of the isolated cells using Western blotting and immunostaining.

The goal of this protocol is to isolate endothelial progenitor cells from umbilical cord blood. Some of the applications include using these cells as a biomarker for identifying patients with cardiovascular risk, treating ischemic diseases, and creating tissue-engineered vascular and heart valve constructs.

Isolamento di cellule endoteliali progenitrici dal sangue del cordone ombelicale umano

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

Imaging Live di Mouse Seconda Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

Dissezione e macchiatura immunofluorescente del corpo di fungo e del fotoricettore neuroni nel cervello adulto Drosophila melanogaster

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the Notch receptor with ligands from neighboring cells induces activation of the signaling pathway (trans-activation), while interaction with ligands from the same cell inhibits signaling (cis-inhibition). Proper balance between trans-activation and cis-inhibition helps establish optimal levels of Notch signaling in some contexts during animal development. Because of the overlapping expression domains of Notch and its ligands in many cell types and the existence of feedback mechanisms, studying the effects of a given post-translational modification on trans- versus cis-interactions of Notch and its ligands in vivo is difficult. Here, we describe a protocol for using Drosophila S2 cells in cell-aggregation assays to assess the effects of knocking down a Notch pathway modifier on the binding of Notch to each ligand in trans and in cis. S2 cells stably or transiently transfected with a Notch-expressing vector are mixed with cells expressing each Notch ligand (S2-Delta or S2-Serrate). Trans-binding between the receptor and ligands results in the formation of heterotypic cell aggregates and is measured in terms of the number of aggregates per mL composed of &#62;6 cells. To examine the inhibitory effect of cis-ligands, S2 cells co-expressing Notch and each ligand are mixed with S2-Delta or S2-Serrate cells and the number of aggregates is quantified as described above. The relative decrease in the number of aggregates due to the presence of cis-ligands provides a measure of cis-ligand-mediated inhibition of trans-binding. These straightforward assays can provide semi-quantitative data on the effects of genetic or pharmacological manipulations on the binding of Notch to its ligands, and can help deciphering the molecular mechanisms underlying the in vivo effects of such manipulations on Notch signaling.

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Complexity of in vivo systems makes it difficult to distinguish between the activation and inhibition of Notch receptor by trans- and cis-ligands, respectively. Here, we present a protocol based on in vitro cell-aggregation assays for qualitative and semi-quantitative evaluation of the binding of Drosophila Notch to trans-ligands vs cis-ligands.

Cella di aggregazione saggi per valutare l'associazione della tacca della drosofila con Trans-ligandi e la sua inibizione di Cis-ligandi

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the ...

Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization

The differentiation of human endometrial stromal cells (HESC) from fibroblast-like appearance into secretory decidua is a transformation required for embryo implantation into the uterine lining of the maternal womb. Improper decidualization has been established as a root cause for implantation failure and subsequent early embryo miscarriage. Therefore, understanding the molecular mechanisms underlying decidualization is advantageous to improving the rate of successful births. In vivo based studies of artificial decidualization are often limiting due to ethical dilemmas associated with human research, as well as translational complications within animal models. As a result, in vitro assays through primary cell culture are often utilized to explore the modulation of decidualization via hormones. This study provides a detailed protocol for the isolation of HESC and subsequent artificial decidualization via the supplementation of hormones to the culturing medium. Further, this study provides a well-designed method to knockdown any gene of interest by utilizing lipid-based siRNA transfections. This protocol permits the optimization of culture purity as well as product yield, thereby maximizing the ability to utilize this model as a reliable method to understand the molecular mechanisms underlying decidualization, and the subsequent quantification of secreted agents by decidualized endometrial stromal cells.

Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization

This study presents a validated and optimized procedure for the isolation and culture of human endometrial stromal cells to conduct in vitro decidualization assay. Further, this study provides a detailed method to efficiently knockdown a specific gene using siRNAs in human endometrial stromal cells.

Isolamento delle cellule stromali dell'endometrio umane per In Vitro decidualizzazione

The differentiation of human endometrial stromal cells (HESC) from fibroblast-like appearance into secretory decidua is a transformation required for ...

Imaging and Analysis of Tissue Orientation and Growth Dynamics in the Developing Drosophila Epithelia During Pupal Stages

Within multicellular organisms, mature tissues and organs display high degrees of order in the spatial arrangements of their constituent cells. A remarkable example is given by sensory epithelia, where cells of the same or distinct identities are brought together via cell-cell adhesion showing highly organized planar patterns. Cells align to one another in the same direction and display equivalent polarity over large distances. This organization of the mature epithelia is established over the course of morphogenesis. To understand how the planar arrangement of the mature epithelia is achieved, it is crucial to track cell orientation and growth dynamics with high spatiotemporal fidelity during development in vivo. Robust analytical tools are also essential to identify and characterize local-to-global transitions. The Drosophila pupa is an ideal system to evaluate oriented cell shape changes underlying epithelial morphogenesis. The pupal developing epithelium constitutes the external surface of the immobile body, allowing long-term imaging of intact animals. The protocol described here is designed to image and analyze cell behaviors at both global and local levels in the pupal abdominal epidermis as it grows. The methodology described can be easily adapted to the imaging of cell behaviors at other developmental stages, tissues, subcellular structures, or model organisms.

Imaging and Analysis of Tissue Orientation and Growth Dynamics in the Developing Drosophila Epithelia During Pupal Stages

This protocol is designed for the imaging and analysis of the dynamics of cell orientation and tissue growth in the Drosophila abdominal epithelia as the fruit fly undergoes metamorphosis. The methodology described here can be applied to the study of different developmental stages, tissues, and subcellular structures in Drosophila or other model organisms.

Imaging e analisi dell'orientamento dei tessuti e delle dinamiche di crescita nell'epiteliale della Drosophila in via di sviluppo durante le fasi pupali

Within multicellular organisms, mature tissues and organs display high degrees of order in the spatial arrangements of their constituent cells. A ...

Generation of a Human iPSC-Based Blood-Brain Barrier Chip

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the blood that can disrupt delicate brain function. As such, the BBB is a major obstacle to the delivery of therapeutics to the CNS. Accumulating evidence suggests that the BBB plays a key role in the onset and progression of neurological diseases. Thus, there is a tremendous need for a BBB model that can predict penetration of CNS-targeted drugs as well as elucidate the BBB&#39;s role in health and disease.
We have recently combined organ-on-chip and induced pluripotent stem cell (iPSC) technologies to generate a BBB chip fully personalized to humans. This novel platform displays cellular, molecular, and physiological properties that are suitable for the prediction of drug and molecule transport across the human BBB. Furthermore, using patient-specific BBB chips, we have generated models of neurological disease and demonstrated the potential for personalized predictive medicine applications. Provided here is a detailed protocol demonstrating how to generate iPSC-derived BBB chips, beginning with differentiation of iPSC-derived brain microvascular endothelial cells (iBMECs) and resulting in mixed neural cultures containing neural progenitors, differentiated neurons, and astrocytes. Also described is a procedure for seeding cells into the organ chip and culturing of the BBB chips under controlled laminar flow. Lastly, detailed descriptions of BBB chip analyses are provided, including paracellular permeability assays for assessing drug and molecule permeability as well as immunocytochemical methods for determining the composition of cell types within the chip.

The blood-brain barrier (BBB) is a multicellular neurovascular unit tightly regulating brain homeostasis. By combining human iPSCs and organ-on-chip technologies, we have generated a personalized BBB chip, suitable for disease modeling and CNS drug penetrability predictions. A detailed protocol is described for the generation and operation of the BBB chip.

Generazione di un chip di barriera emato-encefalica basata sull'iPSC umano

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the ...