Name: preparation of rat serum suitable for mammalian whole embryo culture
Uploaded: 2014-08-03T22:00:00.000+00:00
Duration: 11 min 48 s
Description: Watch this Scientific Journal Video about Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture at JoVE.com

We thank Mr. Hajime Ichijo for video recording and helpful advice concerning editing the video. We also thank the Osumi lab members for animal care. This work is supported by a Grant-in-Aid for Scientific Research on Innovative Area, Neural Diversity and Neocortical Organization, from MEXT of Japan (to N. O.). T. K. was supported by a Research Fellowship of JSPS for Young Scientists.

NOTA: Gli esperimenti sugli animali sono stati condotti in conformità con il National Institutes of Health Guide per la cura e l&#39;uso di animali da laboratorio. Il Comitato per la Sperimentazione Animale della Facoltà di Medicina di Tohoku University ha approvato le procedure sperimentali qui descritte. 1. Anestesia e laparotomia <ol style="margin-left: 40px;"><li> Per la raccolta di sangue, utilizzare specifici ratti maschi Sprague-Dawley esenti da organismi patogeni. Digiunare i ratti per almeno 18 ore, fornendo acqua. Utilizzare pensionati ratti allevatore maschi a 6-8 mesi di età (550-650 g) per la raccolta di sangue. </li><li> Portata 2.5-4.0% isoflurano, che è un anestetico inalatorio, in una scatola collegata ad un apparecchio anestesia introdurre anestesia a 2,0-3,5 L / min per 10 min. Trasferire i ratti nella casella di introdurre l&#39;anestesia. <ol><li> Conferma anesthetization controllando la perdita della risposta alla stimolazione delle zampe con pinze. Coprire il naso di ratti wi° Una maschera collegata ad un apparecchio di anestesia per anestetizzare profondamente i ratti durante il prelievo. La concentrazione e flusso sono mantenuti a 2.5-4.0% e 2.0-3.5 L / min, rispettivamente. NOTA: alotano non deve essere utilizzato per l&#39;anestesia per inalazione perché embrioni coltivati ​​nel siero che vengono raccolti da ratti anestetizzati con questa mostra reagente un ritardo nello sviluppo embrionale rispetto a embrioni coltivati ​​nel siero ottenuti da ratti anestetizzati con isoflurano 22. </li></ol></li><li> Per evitare la contaminazione del pelo, disinfettare la pelle versando il 70% di etanolo sul ventre di ratto anestetizzato. Sollevare la pelle disinfettata e parete addominale in corrispondenza della zona inferiore con un paio di pinze e tagliare questi strati contemporaneamente verso la regione torace con un paio di forbici grandi per esporre gli organi interni. Riflettere l&#39;intestino di fuori della cavità addominale con un paio di pinze. </li><li> Tagliare la pelle e la parete addominale verso gli arti posteriori conpaio di forbici grandi per esporre ulteriormente la regione addominale posteriore. Riflettere il grasso extra al di fuori della cavità addominale con un paio di pinze. </li><li> Sollevare il grasso viscerale sulla linea mediana utilizzando due coppie di pinze e dividere accuratamente il grasso in una direzione parallela esporre l&#39;aorta addominale. Ripetere questa procedura per dividere il grasso viscerale in una direzione longitudinale di identificare facilmente la biforcazione dell&#39;aorta addominale. Inoltre, l&#39;aorta è pulsatile e può essere distinto dalla vena cava adiacente utilizzando questa caratteristica. </li><li> Sollevare il grasso intorno l&#39;aorta addominale e la biforcazione accuratamente con due coppie di pinze e separare il grasso sulla aorta addominale. </li></ol> 2. Raccolta di sangue <ol style="margin-left: 40px;"><li> Inserire delicatamente la punta di un ago G 21 collegato ad una siringa da 20 ml subito cranialmente alla biforcazione dell&#39;aorta, con il bordo inclinato verso il basso. Tenere la siringa con una solamano, e tirare la siringa lentamente per raccogliere 15 ml di sangue da un ratto maschio. </li><li> Rimuovere l&#39;ago dalla siringa, e versare il sangue in due tubi ghiacciati 10 ml sterili test (Figura 1A). Conservare le provette in ghiaccio fino centrifugazione per ritardare la formazione del coagulo. Ridurre il tempo di centrifugazione mediante centrifugazione diversi tubi in una sola volta. </li><li> Euthanize il ratto anestetizzato da toracotomia e tagliando il cuore o decapitazione. </li></ol> 3. Rat Siero Preparazione <ol style="margin-left: 40px;"><li> Centrifugare il sangue raccolto per 5 minuti a 1200 xg e temperatura ambiente (RT) per separare il sangue in strati superiore e inferiore (Figura 1C). </li><li> Conservare le provette a 4 ° C per 1,5-2 ore per risolvere il coagulo di fibrina. </li><li> Sollevare il coagulo di fibrina nello strato superiore usando un paio di pinze curve, e spremere il coagulo di fibrina per separare il siero. Quindi, premere il coagulo di fibrina con le pinze curve fronte downwards prossimità dell&#39;interfaccia tra i due strati (Figura 1D). </li><li> Ripetere passo 3.3 per staccare ulteriormente il coagulo di fibrina. </li><li> Centrifugare le provette per 5 minuti a 1200 xg e RT (Figura 1E). </li><li> Trasferire accuratamente il siero in una provetta sterile da 15 ml con una pipetta sterile in un armadio flusso d&#39;aria laminare. </li><li> Centrifugare il siero di ratto raccolti per 5 minuti a 1200 xg e RT per eliminare cellule rosse rimanenti. </li><li> Pool attenzione il siero in un nuovo tubo 15 ml sterilizzati utilizzando una pipetta sterile per evitare la contaminazione da globuli rossi residue al fondo della provetta (figura 1F). </li><li> Incubare il siero in un bagno d&#39;acqua a 56 ° C per 30 minuti per inattivare il sistema del complemento. </li><li> Raffreddare le provette di siero di ratto a RT nell&#39;armadio aria a flusso laminare. Il siero deve essere frazionare a 10 ml in provette sterili individuali. Conservare le provette a -20 ° C fino al momento dell&#39;uso (Figura 1H). </li></ol>

<ol>
	<li>New, D. A. T., Copp, A. J., Cockroft, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction.">Introduction.</a> Mammalian Postimplantation Embryos. A Practical Approach. , 1-14 (1990).</li><li>Eto, K., Takakubo, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+development+of+rat+embryos+in+culture+during+the+period+of+craniofacial+morphogenesis.">Improved development of rat embryos in culture during the period of craniofacial morphogenesis.</a> J Craniofac Genet Dev Biol. 5, 351-355 (1985).</li><li>Takahashi, M., Osumi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+method+of+rodent+whole+embryo+culture+using+the+rotator-type+bottle+culture+system.">The method of rodent whole embryo culture using the rotator-type bottle culture system.</a> J Vis Exp. (42), e2170 (2010).</li><li>New, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+embryo+culture,+teratogenesis,+and+the+estimation+of+teratologic+risk.">Whole embryo culture, teratogenesis, and the estimation of teratologic risk.</a> Teratology. 42, 635-642 (1990).</li><li>Matsuo, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mutation+in+the+Pax-6+gene+in+rat+small+eye+is+associated+with+impaired+migration+of+midbrain+crest+cells.">A mutation in the Pax-6 gene in rat small eye is associated with impaired migration of midbrain crest cells.</a> Nat Genet. 3, 299-304 (1993).</li><li>Nomura, T., Holmberg, J., Frisen, J., Osumi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pax6-dependent+boundary+defines+alignment+of+migrating+olfactory+cortex+neurons+via+the+repulsive+activity+of+ephrin+A5.">Pax6-dependent boundary defines alignment of migrating olfactory cortex neurons via the repulsive activity of ephrin A5.</a> Development. 133, 1335-1345 (2006).</li><li>Yamamoto, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nodal+antagonists+regulate+formation+of+the+anteroposterior+axis+of+the+mouse+embryo.">Nodal antagonists regulate formation of the anteroposterior axis of the mouse embryo.</a> Nature. 428, 387-392 (2004).</li><li>Inoue, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+cadherins+in+maintaining+the+compartment+boundary+between+the+cortex+and+striatum+during+development.">Role of cadherins in maintaining the compartment boundary between the cortex and striatum during development.</a> Development. 128, 561-569 (2001).</li><li>Takahashi, M., Osumi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pax6+regulates+specification+of+ventral+neurone+subtypes+in+the+hindbrain+by+establishing+progenitor+domains.">Pax6 regulates specification of ventral neurone subtypes in the hindbrain by establishing progenitor domains.</a> Development. 129, 1327-1338 (2002).</li><li>Calegari, F., Haubensak, W., Yang, D., Huttner, W. B., Buchholz, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-specific+RNA+interference+in+postimplantation+mouse+embryos+with+endoribonuclease-prepared+short+interfering+RNA.">Tissue-specific RNA interference in postimplantation mouse embryos with endoribonuclease-prepared short interfering RNA.</a> Proc Natl Acad Sci U S A. 99, 14236-14240 (2002).</li><li>Takahashi, M., Nomura, T., Osumi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transferring+genes+into+cultured+mammalian+embryos+by+electroporation.">Transferring genes into cultured mammalian embryos by electroporation.</a> Dev Growth Differ. 50, 485-497 (2008).</li><li>Pryor, S. E., Massa, V., Savery, D., Greene, N. D., Copp, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Convergent+extension+analysis+in+mouse+whole+embryo+culture.">Convergent extension analysis in mouse whole embryo culture.</a> Methods Mol Biol. 839, 133-146 (2012).</li><li>Kikkawa, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dmrta1+regulates+proneural+gene+expression+downstream+of+Pax6+in+the+mammalian+telencephalon.">Dmrta1 regulates proneural gene expression downstream of Pax6 in the mammalian telencephalon.</a> Genes Cells. 18, 636-649 (2013).</li><li>Tabata, H., Nakajima, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+in+utero+gene+transfer+system+to+the+developing+mouse+brain+using+electroporation:+visualization+of+neuronal+migration+in+the+developing+cortex.">Efficient in utero gene transfer system to the developing mouse brain using electroporation: visualization of neuronal migration in the developing cortex.</a> Neuroscience. 103, 865-872 (2001).</li><li>Fukuchi-Shimogori, T., Grove, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neocortex+patterning+by+the+secreted+signaling+molecule+FGF8.">Neocortex patterning by the secreted signaling molecule FGF8.</a> Science. 294, 1071-1074 (2001).</li><li>Saito, T., Nakatsuji, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+gene+transfer+into+the+embryonic+mouse+brain+using+in+vivo+electroporation.">Efficient gene transfer into the embryonic mouse brain using in vivo electroporation.</a> Dev Biol. 240, 237-246 (2001).</li><li>Turnbull, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasound+backscatter+microscopy+of+mouse+embryos.">Ultrasound backscatter microscopy of mouse embryos.</a> Methods Mol Biol. 135, 235-243 (2000).</li><li>Pierfelice, T. J., Gaiano, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasound-guided+microinjection+into+the+mouse+forebrain+in+utero+at+E9.5.">Ultrasound-guided microinjection into the mouse forebrain in utero at E9.5.</a> J Vis Exp. (45), e2047 (2010).</li><li>Quinlan, G. A., Khoo, P. L., Wong, N., Trainor, P. A., Tam, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+grafting+and+labeling+in+postimplantation+mouse+embryos.">Cell grafting and labeling in postimplantation mouse embryos.</a> Methods Mol Biol. 461, 47-70 (2008).</li><li>Nagy, A., Gertsenstein, M., Vinterstein, K., Behringer, R., Nagy, A., Gertsenstein, M., Vinterstein, K., Behringer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roller+culture+of+postimplantation+embryos.">Roller culture of postimplantation embryos.</a> Manipulating the Mouse Embryo: A Laboratory Manual. , 237-241 (2003).</li><li>Garcia, M. D., Udan, R. S., Hadjantonakis, A. K., Dickinson, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+rat+serum+for+culturing+mouse+embryos.">Preparation of rat serum for culturing mouse embryos.</a> Cold Spring Harb Protoc. 2011, 391-393 (2011).</li><li>Brown-Woodman, P. D., Ritchie, H. E., Korabelnikoff, A., Emmanuel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replacement+of+ether+with+alternate+volatile+anesthetics+for+collection+of+rat+serum+used+in+embryo+culture.">Replacement of ether with alternate volatile anesthetics for collection of rat serum used in embryo culture.</a> Toxicol In Vitro. 18, 719-724 (2004).</li><li>Gray, J., Ross, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+tube+closure+in+mouse+whole+embryo+culture.">Neural tube closure in mouse whole embryo culture.</a> J Vis Exp. (56), e3132 (2011).</li><li>Machado, C. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstruction+of+phrenic+neuron+identity+in+embryonic+stem+cell-derived+motor+neurons.">Reconstruction of phrenic neuron identity in embryonic stem cell-derived motor neurons.</a> Development. 141, 784-794 (2014).</li><li>Glanville-Jones, H. C., Woo, N., Arkell, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successful+whole+embryo+culture+with+commercially+available+reagents.">Successful whole embryo culture with commercially available reagents.</a> Int J Dev Biol. 57, 61-67 (2013).</li><li>Ornoy, A., Yacobi, S., Yaffee, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+of+culture+of+11.5-day-old+rat+embryos+in+DMEM/F12+and+20%+fetal+bovine+serum.">A simple method of culture of 11.5-day-old rat embryos in DMEM/F12 and 20% fetal bovine serum.</a> J Anat. 203, 419-423 (2003).</li><li>Moore-Scott, B. A., Gordon, J., Blackburn, C. C., Condie, B. G., Manley, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+serum-free+in+vitro+culture+technique+for+midgestation+mouse+embryos.">New serum-free in vitro culture technique for midgestation mouse embryos.</a> Genesis. 35, 164-168 (2003).</li><li>Kalaskar, V. K., Lauderdale, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+Embryonic+Development+in+a+Serum-free+Whole+Embryo+Culture+System.">Mouse Embryonic Development in a Serum-free Whole Embryo Culture System.</a> J Vis Exp. (85), e50308 (2014).</li></ol>

No conflicts of interest are declared.

Risultati riproducibili in esperimenti WEC dipendono l&#39;uso di siero di ratto IC alta qualità e accurata tecnica embrione dissezione 3. Non accorciare il periodo di digiuno prima del prelievo del sangue, perché il digiuno è necessario standardizzare le concentrazioni di glucosio nel sangue. Ratti femmina non devono essere utilizzati per la preparazione di siero perché i livelli ormonali cambiano in femmine secondo il ciclo estrale, e tali cambiamenti di ormoni estradiolo non sono adatti per colture emb...

Una varietà di modelli animali sono utilizzati in biologia dello sviluppo per studiare meccanismi di sviluppo a livello molecolare e cellulare. Ad esempio, anfibi e aviarie sono stati ampiamente usati come animali modello classico che sono adatti per la manipolazione diretta di embrioni perché questi embrioni si sviluppano al di fuori della madre. In contrasto con questi animali, embrioni mammiferi crescono nell&#39;utero della madre, e la crescita in fasi successive è strettamente dipendente dalla funzione dell&#39;utero. Pertanto, è tipicamente difficile da manipolare direttamente embrioni di mammifero come quelli di topo e nel ratto nelle fasi iniziali. Nel 1960, Denis New istituito un mammifero intera coltura degli embrioni (WEC), tecnica che utilizza apparecchiature WEC con un apporto di ossigeno continuo e controllo di calore 1. In WEC, topo e ratto embrioni possono crescere ex vivo (cioè, al di fuori dell&#39;utero). Anche se la tecnica del WEC è stato spesso utilizzato in teratologia con l&#39;aggiunta di vari chemicAl composti nel terreno di coltura, questa tecnica è stata utilizzata anche in diversi studi di biologia dello sviluppo per esaminare unici meccanismi di sviluppo nei mammiferi 2-4. Ad esempio, WEC è combinata con altre tecniche, quali l&#39;etichettatura delle cellule, in wild-type e mutanti embrioni utilizzando colorante fluorescente 5, il trapianto di cellule 6, e l&#39;introduzione del gene via lipofezione 7 ed elettroporazione 8-13. Recentemente, in manipolazione utero è stato utilizzato per analizzare i processi di sviluppo in embrioni di roditori in fasi successive ed è stato combinato con tecniche di elettroporazione 14-16. Tuttavia, queste tecniche non sono adatti per la manipolazione di postimpianto e midgestation embrioni causa di difficoltà a raggiungere iniezione locale accurata della soluzione di DNA in embrioni nelle fasi iniziali. Sebbene il trapianto di cellule eco-guidata e l&#39;iniezione di vettori virali in embrioni precoci (cioè, Sono stati segnalati E8.5-E-9.5 nel topo) in utero precedentemente 17,18, ottime capacità sono necessarie per eseguire questi esperimenti con un alto tasso di successo. Pertanto, WEC con alta accessibilità ex vivo ha dei vantaggi rispetto alla manipolazione di topo e ratto embrioni. Subito centrifugati (IC) siero di ratto preparati da ratti maschi è spesso usato per le medie WEC. Quando gli embrioni sono coltivate nella fase post-impianto (cioè, prima del E8.0 nel topo o E10.0 nel ratto), una miscela di terreno sintetico e siero di ratto IC viene spesso usata come mezzo per WEC 19. Tuttavia, per gli embrioni di cultura a metà della gestazione (cioè., E8.0-12.5 in embrioni di topo o E10.0-E14.5 in embrioni di ratto), siero al 100% dovrebbe essere utilizzato come mezzo perché nessun media alternativi attualmente disponibili consentono embrioni a crescere normalmente in vitro per più di 2 giorni. La preparazione del siero di ratto di alta qualità èun passo fondamentale per il raggiungimento riproducibilità negli esperimenti WEC. Rispetto a centrifugazione differite, IC ha il vantaggio di ridurre emolisi quando il siero viene raccolto comprimendo il coagulo di fibrina perché la maggior parte dei globuli rossi sono stati già separato dal coagulo di fibrina. Come siero di ratto emolitica non riesce a sostenere la crescita normale di ratto e di topo embrioni, preparazione del siero utilizzando immediata centrifugazione è preferibile preparato utilizzando centrifugazione ritardato. Il nostro protocollo contiene due passaggi aggiuntivi rispetto ad altri protocolli 18-20 (es., Conservare il sangue raccolto in ghiaccio prima della prima centrifugazione e mantenere i campioni di sangue raccolti per 2 ore a 4 ° C dopo la prima centrifugazione). Il primo passo può ritardare la formazione del coagulo di sangue, e quest&#39;ultimo passo promuove la solidificazione del coagulo di fibrina per facile spremitura. Pertanto, il nostro protocollo può essere utilizzato da principianti. Tuttavia, la riproduzione di raccolta del sangue e sieroestrazione con precisione semplicemente facendo riferimento ai libri di protocollo è estremamente difficile 19-21. In questo articolo video, dimostriamo il nostro protocollo standard per la preparazione di siero ottimale ratto IC, che comprende la raccolta di sangue dall&#39;aorta addominale di ratti maschi e estrazione del siero mediante centrifugazione.

<table><tbody><tr><td>Isoflurane</td><td>Abbott</td><td>B506</td><td>For rat anesthesia.</td></tr><tr><td>Large scissors</td><td>Napox</td><td>B-7H</td><td>Stelized scissors for cutting the skin and muscle of rats.&amp;nbsp;</td></tr><tr><td>Curved forceps</td><td>Napox</td><td>A-3-2</td><td>Stelized forceps for picking up the skin of rats and squeezing the fibrin clot.</td></tr><tr><td>Sprague-Dawley (SD) rat</td><td>Charles Rivers Laboratories</td><td></td><td>Retired male rats from colony in the lab or purchased retired male rats.</td></tr><tr><td>Syringe (20 ml)</td><td>TERUMO</td><td>SS-29ESZ</td><td></td></tr><tr><td>Needle (21 G x 5/8&quot;)</td><td>TERUMO</td><td>NN-2116R</td><td></td></tr><tr><td>Sterile test (spitz) tube (10 ml)</td><td>ASIAKIZAI</td><td>1101C000B-10</td><td>For collection of boold</td></tr><tr><td>Sterile disposable pipette</td><td>Eiken Chemical</td><td>CD2000 No.4</td><td></td></tr><tr><td>Sterilie disposable tube (15 ml)&amp;nbsp;</td><td>Falcon</td><td>2196</td><td></td></tr></tbody></table>

preparation of rat serum suitable for mammalian whole embryo culture

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for investigating the mechanisms of developmental processes. Immediately centrifuged (IC) rat serum is commonly used for WEC and is essential for the growth and development of cultured mouse and rat embryos ex vivo. For the culture of midgestation embryos (i.e., E8.0-12.5 for the mouse, and E10.0-14.5 for the rat), 100% rat serum is the best media for supporting the growth of the embryo ex vivo. To prepare rat serum suitable for WEC, the collected blood should be centrifuged immediately to separate the blood cells from the plasma fraction. After centrifugation, the fibrin clot forms in the upper layer; this clot should be squeezed gently using a pair of sterile forceps and subsequently centrifuged to completely separate the blood cells from the serum. In this video article, we demonstrate our standard protocol for the preparation of optimal IC rat serum, including blood collection from the abdominal aorta of male rats and extraction of the serum by centrifugation.

La Figura 1 mostra i risultati rappresentativi delle procedure descritte per la separazione delle cellule del sangue e siero. Abbiamo tipicamente ottiene 15 ml di sangue da un ratto maschio, in pensione (Figura 1A). Per centrifugazione, il sangue raccolto può essere separato in uno strato superiore contenente siero e il coagulo di fibrina, che è indicato con una freccia, e uno strato inferiore contenente i globuli (Figura 1C). È importante sottolineare che i campioni...

Watch this Scientific Journal Video about Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture at JoVE.com

Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture

Mammalian whole embryo culture (WEC) is widely used in teratology and developmental biology. Immediately centrifuged rat serum is commonly provided as a medium for both mouse and rat WEC. In this video, we demonstrate our standard protocol for the preparation of high-quality rat serum suitable for mammalian WEC.

Preparazione di Rat Siero Adatto per mammiferi tutta la cultura Embrione

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for ...

preparation-of-rat-serum-suitable-for-mammalian-whole-embryo-culture

Research

JoVE Journal

Biology

13.8K Views.  Jichi Medical University. Mammalian whole embryo culture (WEC) is widely used in teratology and developmental biology. Immediately centrifuged rat serum is commonly provided as a medium for both mouse and rat WEC. In this video, we demonstrate our standard protocol for the preparation of high-quality rat serum suitable for mammalian WEC.

Video: Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein, tandem dimer Tomato (tdTomato), are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons, evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations.

A protocol to generate a co-culture system consisting of neurons derived from induced pluripotent stem cells (iPSCs), primary cortical neurons and astrocytes is described. This co-culture system allows detection of the formation of synaptic contacts and circuits between new, iPSC-derived neurons and pre-existing cortical neurons expressing channelrhodopsin-2.

Un approccio per la valutazione optogenetic Formazione di connessioni neuronali in un sistema di co-coltura

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are ...

Ricerca

Biologia dello sviluppo

Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as well as the generation of mature neural cell types for further study. In this protocol we describe a method for the differentiation of ESC to neural progenitors using serum-free, monolayer culture. The method is scalable, efficient and results in production of ~70% neural progenitor cells within 4 - 6 days. It can be applied to ESC from various strains grown under a variety of conditions. Neural progenitors can be allowed to differentiate further into functional neurons and glia or analyzed by microscopy, flow cytometry or molecular techniques. The differentiation process is amenable to time-lapse microscopy and can be combined with the use of reporter lines to monitor the neural specification process. We provide detailed instructions on media preparation and cell density optimization to allow the process to be applied to most ESC lines and a variety of cell culture vessels.

This protocol describes in detail the method for generating neural progenitors from embryonic stem cells using a serum-free monolayer method. These progenitors can be used to derive mature neural cell types or to study the process of neural specification and is amenable to multiwell format scaling for compound screening.

Differenziamento neurale delle cellule staminali embrionali di topo in siero privo monostrato Cultura

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as ...

Methods for Precisely Localized Transfer of Cells or DNA into Early Postimplantation Mouse Embryos

Manipulation and culture of early mouse embryos is a powerful yet largely under-utilized technology enhancing the value of this model system. Conversely, cell culture has been widely used in developmental biology studies. However, it is important to determine whether in vitro cultured cells truly represent in vivo cell types. Grafting cells into embryos, followed by an assessment of their contribution during development is a useful method to determine the potential of in vitro cultured cells. In this study, we describe a method for grafting cells into a defined site of early postimplantation mouse embryos, followed by ex vivo culture. We also introduce an optimized electroporation method that uses glass capillaries of known diameter, allowing precise localization and adjustment of the number of cells receiving exogenous DNA with both high transfection efficiency and low cell death. These techniques, which do not require any specialized equipment, render experimental manipulations of the gastrulation and early organogenesis-stage mouse embryo possible, allowing analysis of commitment in cultured cell subpopulations and the effect of genetic manipulations in situ on cell differentiation.

We demonstrate a method for grafting cultured cells into defined sites of early mouse embryos to determine their in vivo potential. We also introduce an optimized electroporation method that uses glass capillaries of known diameter, allowing the precise delivery of exogenous DNA into a few cells in the embryos.

Metodi di trasferimento Proprio localizzata di cellule o DNA in precoce post-impianto di embrioni di topo

Manipulation and culture of early mouse embryos is a powerful yet largely under-utilized technology enhancing the value of this model system. Conversely, ...

Preparation of Rat Oligodendrocyte Progenitor Cultures and Quantification of Oligodendrogenesis Using Dual-infrared Fluorescence Scanning

Efficient oligodendrogenesis is the therapeutic goal of a number of areas of research including spinal cord injury, neonatal hypoxia, and demyelinating diseases such as multiple sclerosis and transverse myelitis. Myelination is required to not only facilitate rapid impulse propagation within the central nervous system, but also to provide trophic support to underlying axons. Oligodendrocyte progenitor cells (OPCs) can be studied in vitro to help identify factors that may promote or inhibit oligodendrocyte differentiation. To date, many of the methods available to evaluate this process have either required large numbers of cells, thus limiting the number of conditions that can be investigated at any one time, or labor-intensive methods of quantification. Herein, we describe a protocol for the isolation of large numbers of highly pure OPCs together with a fast and reliable method to determine oligodendrogenesis from multiple conditions simultaneously. OPCs are isolated from P5-P7 neonatal rat cortices and grown in vitro for three days prior to differentiation. Four days after differentiation, oligodendrogenesis is evaluated using a dual-infrared fluorescence-scanning assay to determine expression of the myelin protein.

Oligodendrocytes are the myelinating cells of the central nervous system. This protocol describes a method for the isolation and culture of their precursors, oligodendrocyte progenitor cells, from rat cortices, as well as a fast and reliable quantitative method to evaluate oligodendrogenesis in vitro in response to experimental factors.

Preparazione delle Culture Rat progenitrici degli oligodendrociti e la quantificazione dei Oligodendrogenesis Utilizzando Dual-infrarossi fluorescenza Scansione

Efficient oligodendrogenesis is the therapeutic goal of a number of areas of research including spinal cord injury, neonatal hypoxia, and demyelinating ...

Isolation of Murine Embryonic Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level.

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

Isolamento di cellule murine embrionali Hemogenic endoteliali

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied successfully to the isolation of mouse and human primary neurons and neural progenitors. Dissociated neurons are maintained in serum-free medium up to several weeks. These cultures can be used for nucleofection, immunocytochemistry, nucleic acids preparation, as well as electrophysiology. Older neuronal cultures can also be transfected with a good efficiency rate by lentiviral transduction and, less efficiently, with calcium phosphate or lipid-based methods such as lipofectamine.

We describe a rapid methodology to isolate and culture hippocampal and cortical neurons from rodent embryos. This protocol allows us to perform experiments in which nearly pure neuronal cultures are required.

Isolamento e la coltura di cellule embrionali neuronali di ratto: un protocollo veloce

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied ...

Neuroscienze

Preparation of Rat Brain Aggregate Cultures for Neuron and Glia Development Studies

An in vitro system that recapitulates the development and differentiation of progenitors into mature neurons and glia in the central nervous system (CNS) would provide a powerful platform for neuroscientists to investigate axo-glial interactions, properties and differentiation of multipotent progenitors, and progression of oligodendroglial lineage cells at the cellular and molecular level.  We describe here a CNS aggregate culture system from embryonic rat forebrains, which can be maintained in a serum-free medium up to 3-4 weeks and is used in our laboratory as a model to study neuron-glia interaction and CNS myelination.  This video clip will demonstrate how to isolate and grow these CNS aggregate cultures from E16 rat brain.  Furthermore, from the same brain dissection, highly enriched regular dissociated neuronal cultures can be readily obtained and used for various studies on CNS neurons or used for co-cultures with other cells. 

A protocols for an embryonic rat brain aggregate culture system is described. Multipotent progenitors in the aggregates can develop and differentiate into neurons, astrocytes and oligodendrocytes.

Preparazione delle Culture del cervello di ratto aggregati per Neuron e Studi sullo Sviluppo Glia

An in vitro system that recapitulates the development and differentiation of progenitors into mature neurons and glia in the central nervous system (CNS) ...

Biologia

Ex Utero Culture of Mouse Embryos from Pregastrulation to Advanced Organogenesis

Postimplantation mammalian embryo culture methods have been generally inefficient and limited to brief periods after dissection out of the uterus. Platforms have been recently developed for highly robust and prolonged ex utero culture of mouse embryos from egg-cylinder stages until advanced organogenesis. These platforms enable appropriate and faithful development of pregastrulating embryos (E5.5) until the hind limb formation stage (E11). Late gastrulating embryos (E7.5) are grown in rotating bottles in these settings, while extended culture from pregastrulation stages (E5.5 or E6.5) requires a combination of static and rotating bottle cultures. In addition, sensitive regulation of O2 and CO2 concentration, gas pressure, glucose levels, and the use of a specific ex utero culture medium are critical for proper embryo development. Here, a detailed step-by-step protocol for extended ex utero mouse embryo culture is provided. The ability to grow normal mouse embryos ex utero from gastrulation to organogenesis represents a valuable tool for characterizing the effect of different experimental perturbations during embryonic development.

Ex Utero Culture of Mouse Embryos from Pregastrulation to Advanced Organogenesis

An enhanced platform for whole-embryo culture allows continuous and robust ex utero development of postimplantation mouse embryos for up to six days, from pregastrulation stages until advanced organogenesis. In this protocol, we detail the standard procedure for successful embryo culture using static plates and rotating bottle systems.

Ex Utero Coltura di embrioni di topo dalla pregastrulazione all'organogenesi avanzata

Postimplantation mammalian embryo culture methods have been generally inefficient and limited to brief periods after dissection out of the uterus. ...

The Method of Rodent Whole Embryo Culture using the Rotator-type Bottle Culture System

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development and growth of mammalian embryos are critically dependent on the function of the placenta, WEC technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages during embryonic day (E) 6.5-E12.5 in the mouse or E8.5-E14.5 in the rat 2, 3, 4. In WEC, we can directly target desired areas of embryos using fine glass capillaries because embryos can be manipulated under the microscope. Therefore, rodent WEC is very useful technique when we want to study dynamic developmental processes of postimplanted mammalian embryos. Up to date, several types of WEC systems have been developed 1. Among those, the rotator-type bottle culture system is most popular and suitable for long-term culture of embryos at midgestation, i.e., after E9.5 and E11.5 in the mouse and rat, respectively 1. In this video protocol, we demonstrate our standard procedures of rat WEC after E12.5 using a refined model of the original rotator system, which was designed by New and Cockroft 5, 6, and introduce various applications of WEC technique for studies in mammalian developmental biology.

Whole embryo culture technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages. In this video protocol, we demonstrate our standard procedures of rat whole embryo culture after E12.5 using the rotator-type bottle culture system.

Il metodo di coltura degli embrioni roditori intero utilizzando il Rotator tipo di sistema cultura Bottiglia

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development ...

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O2 / 5% CO2 in a rolling bottle culture apparatus at 37 &#176;&#8203;C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.

Serum utilized in embryo cultures contains unknown components that can affect the outcome of experiments especially in studies involving signaling interactions. Here we utilized a serum-free oxygenated culture system and show that mid-gestation mouse embryos cultured for 16-40 hr&#160;exhibit morphological development comparable to embryos developing in utero.

Embrionali di topo di sviluppo in un embrione di tutta la cultura di sistema privo di siero

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in ...