Vorremmo ringraziare Ryan Maneri (Oystercatcher Productions, LLC) per le riprese la nostra procedura e Rodney McPhail (Dipartimento di Scienze Biologiche, Purdue University) per l&#39;assistenza con l&#39;editing del video dissezione. La ricerca nel laboratorio di Suter è sostenuto da finanziamenti del NIH (R01 NS049233) e il Centro Bindley Bioscience alla Purdue University di DMS

Soluzioni <ul><li> L15-ASW terreno di coltura cellulare (1l) <ul><li> 1 bustina di polvere L15 </li><li> aggiungere 800 ml di H 2 O ultrapura </li><li> NaCl 400 mM </li><li> MgSO 4 27 mM </li><li> MgCl 2 28 mM </li><li> L-Glutammina 4 mM </li><li> Gentamicina 50 mg / ml </li><li> HEPES 5 mM </li><li> Regolare a pH 7,9 </li><li> Aggiungere goccia a goccia CaCl 2 9,3 mm (arresto se precipitati) </li><li> Aggiungi H 2 O ultrapura a 1 l </li><li> Controllare osmolarità (950-1000 mg / kg) con osmometer (pressione di vapore, Wescor # 5520) </li><li> Filtro 0,22 micron con una positiva unità di filtrazione a pressione (Filtri: SVGV010RS Millipore) </li><li> Conservare a 4 ° C fino a 1 mese </li></ul></li><li> Poli-L-lisina (70-150 kD) 200 mg / ml di soluzione in magazzino sterile H 2 O ultrapura conservato a -20 ° C </li><li> 0,5 M soluzione di MgCl 2 </li></ul> 1 ° giorno: dissezione di Aplysia ganglio addominale Preparazione della soluzione enzimatica dissociazione <ol><li> Pesare 9-10 mg di dispasi enzima (Worthington, Cat: LS02111) in una provetta Eppendorf </li><li> Aggiungere 900 ml di L15-ASW terreno di coltura, e 100 ml di H 2 O sterile ultrapura </li><li> Mescolare e posto a temperatura ambiente (RT) </li></ol> Dissezione di ganglio addominale <ol><li> Riempire una siringa da 50 ml con 0,5 M MgCl 2 soluzione. </li><li> Prendere un animale da serbatoio, trattata con delicatezza per evitare la difesa dell&#39;animale risposta inchiostrazione. </li><li> Posizionare il Aplysia su un lato su una tavola di dissezione (Styrofoam bordo), parte rostrale a destra, lato caudale a sinistra. </li><li> Inserire l&#39;ago nel animale appena dietro la testa e iniettare tutta MgCl 2 soluzione nella cavità del corpo. </li><li> Strofinare delicatamente l&#39;animale per disperdere i MgCl 2 soluzione tutto cavità del corpo, attendere qualche minuto per l&#39;animale di essere completamente anestetizzata. </li><li> Attacca la testa e la coda dell&#39;animale alla scheda di dissezione con due aghi. </li><li> Usare pinze per sollevare la pelle, tagliare la pelle e lo strato muscolare con le forbici grandi verso la testa e la coda per fare una grande apertura sul lato. </li><li> Trova il ganglio addominale sotto il pennello di tipo ovaio. Usare pinze per tenere i nervi di collegamento di circa 1 cm di fronte al ganglio addominale, tagliare i nervi con le forbici dissezione di fronte alla posizione Pinza e dietro il ganglio addominale. </li><li> Ganglio posto nella soluzione dispasi e incubare per 15-16 ore a 22 ° C con una temperatura controllata bagnomaria. </li></ol> Giorno 2: Placcatura di cellule borsa Aplysia Preparazione poli-lisina rivestite coprioggetto <ol><li> In un banco di flusso, 35 millimetri preparare tre piatti di Petri come lavorare piatti per la dissezione del ganglio, riempiendole di 4 ml L15-ASW media ciascuno. Trasferire il ganglio addominale dalla soluzione dispasi a uno dei piatti di funzionare dopo 15,5 ore di digestione. </li><li> Utilizzando pinze prendere acido-pulito # 1.5 coprioggetti (22x22 mm) conservati in etanolo, rimuovere l&#39;alcol e sterilizzare coprioggetto da fiamme brevemente su becco Bunsen. Lasciateli raffreddare prima di mettere brevemente i coprioggetti in capsule di Petri. </li><li> Preparare una punta ricurva placcatura piegando una punta di giallo sopra il becco Bunsen (fiamma solo per breve tempo per evitare la fusione della plastica!). </li><li> Preparare la quantità appropriata di 20 mg / ml soluzione di poli-lisina diluendo 200 mg / ml Soluzione sterile con H 2 O ultrapura. </li><li> Coprire ogni coprioggetto con 500 ml di 20 mg / ml di poli-lisina e incubare per 20 minuti a RT. </li><li> Lavare ogni coprioggetto 3-5 volte con 500 microlitri sterile H 2 O ultrapura ciascuno. </li><li> Rimuovere l&#39;acqua dal coprioggetto; riempire ogni piatto con 4 ml di L15-ASW terreno di coltura. </li></ol> Dissociazione delle cellule borsa <ol><li> Sotto un microscopio dissezione, tagliare il ganglio addominale a metà forbici e pinze con dissezione precedentemente puliti con etanolo al 95% (Figura 1 linea 1). </li><li> Su una metà del ganglio, tagliare tra cluster borsa cellulare e le cellule hemiganglion (Figura 1 linea 2), e tagliare via le estensioni dei nervi rimasti al di là di cellule borsa (Figura 1 linea 3). Lavorare su una metà del ganglio alla volta, lasciando l&#39;altra metà nel piatto, mentre le cellule dal primo gruppo sono stati placcati. </li><li> Utilizzando due pinze (o una pinza e forbici dissezione), sacco a grappolo rilascio delle cellule mettendo da parte la guaina di tessuto connettivo che circonda il cluster di cellule borsa. Tagliare il tessuto connettivo con le forbici dissezione, se necessario. Attenzione a non stringere e toccare il cluster di cellule borsa troppo durante questo processo. </li><li> Quando la maggior parte del tessuto connettivo viene rimosso, trasferire il bgrappolo di cellule ag in un piatto nuovo a lavorare con la punta piegata giallo preparato in precedenza. Fare attenzione a non lasciare che il gruppo di cellule sacchetto essere intrappolato dentro la punta di colore giallo o bloccati in aria / medio interfaccia. </li><li> Triturare il cluster con la punta giallo per rimuovere le cellule singolo sacchetto. Fate questo pipettando il cluster dentro e fuori la punta. Iniziare con basse forze di taglio e aumentare la forza se necessario. Utilizzare sempre la più bassa forza necessaria per rimuovere i neuroni senza uccidere le cellule troppi. </li><li> Raccogliere 3-5 cellule sane borsa e trasferirli in un piatto con un poli-lisina rivestite coprioggetto. Evitate di raccogliere le cellule morte (sono nero / trasparente al centro) e detriti del tessuto connettivo. </li><li> Ripetere il passaggio 6. e luogo circa 15-25 neuroni vivere al centro di ogni coprioggetto. Se le cellule morte e detriti vengono trasferiti anche, rimuovere il materiale indesiderato di raccoglierlo o soffiare via dalle cellule sane. Questo processo richiede circa 2-3 ore per entrambi i gruppi. </li><li> Una volta finito di tenere i piatti della cultura in panchina di flusso per almeno 2 ore a temperatura ambiente per permettere alle cellule di allegare (tenere banco flusso ventilatore spento per evitare vibrazioni indesiderate). </li><li> Porre le capsule in un incubatore a 14 ° C per una notte, o fino a quando un ulteriore uso. Coni di crescita si possono sviluppare in 4-10 ore. </li></ol><img src="/files/ftp_upload/old/662_2008_2_10_0/image001.jpg" alt="Figura 1. Schematica del ganglio Aplysia californica addominale. Linee di colore indicano dove tagliare per ottenere gruppi di cellule borsa." /> Figura 1. Schematica del ganglio Aplysia californica addominale. Linee di colore indicano dove tagliare per ottenere gruppi di cellule borsa. Giorno 3: Impostazione da camera per immagini dal vivo di cellule borsa Aplysia Assemblaggio di immagini da camera <ol><li> Prima di utilizzare cellule borsa per esperimenti dal vivo l&#39;imaging cellulare, ispezionare le cellule utilizzando un microscopio a bassa potenza. Se le cellule non hanno sviluppato coni di crescita sana, di approvvigionamento a medio dolce e lasciarlo a temperatura ambiente per 1-2 ore. </li><li> Per la fornitura di piatti con prodotti freschi di media L15-ASW, rimuovere la metà (~ 2 ml) di media età con una linea di vuoto, e aggiungere 2 ml di nuovi L15-ASW medio per capsula di Petri. E &#39;importante non rimuovere completamente tutti i media dal piatto tutto in una volta, in modo da rimuovere le cellule dal vetrino. </li><li> Per l&#39;imaging cellulare borsa, montiamo due coprioggetto e due distanziatori di plastica in un sandwich-come camera di imaging. Il coprioggetto top nel panino viene tagliato corto per permettere un facile scambio di medie e reagenti. Durante il montaggio, coprioggetto con le cellule borsa allegata devono essere immersi in L15-ASW medio in ogni momento di impedire alle cellule di sloggiare. </li><li> Per preparare la camera, tagliati fuori ~ 2 mm dal lato destro di mm # 1 coprioggetto 22x22 con diamante penna (tagliatore di vetro) e il righello. </li><li> Usando una siringa da 1 ml, applicare grasso alto vuoto ai due lati del vetrino più brevi, e allegare distanziatori di plastica per ogni lato. Ingrassare vuoto nella parte superiore di entrambi i distanziali. </li><li> Invertire il coprioggetto e montaggio distanziatore, e posto in capsula di Petri contenente cellule borsa su un vetrino coprioggetti. Allineare e premere delicatamente verso il basso per attaccare coprioggetto dall&#39;alto verso il basso coprioggetto con la fine inversione di due Q-tips. </li><li> Utilizzando pinze, sollevare delicatamente il montaggio panino fuori della scatola di Petri, senza il supporto fuoriuscire. Allineare le due coprioggetto, se necessario, e pulire medio in eccesso con un Kimwipe. Posizionare il panino sulla parte inferiore della porta vetrino da microscopio e fissare il coperchio con cura utilizzando clip. </li><li> Pulire il centro del lato superiore del panino con Q-tips tamponò in detergente per vetri (Sparkle è usato come detergente per vetri). Rapidamente asciugare il detergente per vetri con un secondo, asciutto Q-tip per evitare linee striscia. Fare lo stesso per il coprioggetto fondo. Coprioggetto pulito, privo di linee striscia sono importanti per l&#39;imaging di alta qualità. </li></ol> Cellule borsa sul microscopio Dal Aplysia colture di cellule neuronali borsa sono a bassa densità, è meglio se il vostro setup microscopio ha funzioni di posizionamento stadio per facilitare il passaggio tra le celle. Cellule borsa può essere facilmente conservato a temperatura ambiente per diverse ore sul palco durante gli esperimenti di imaging. Cambio del mezzo periodicamente per evitare l&#39;evaporazione.

<ol>
	<li>Forscher, P., Kaczmarek, L. K., Buchanan, J. A., Stephen, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+AMP+induces+changes+in+distribution+and+transport+of+organelles+within+growth+cones+of+Aplysia+bag+cell+neurons.">Cyclic AMP induces changes in distribution and transport of organelles within growth cones of Aplysia bag cell neurons.</a> J. Neurosci. 7, 3600-3611 (1987).</li><li>Suter, D. M., Errante, L. D., Belotserkovsky, V., Forscher, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Ig+superfamily+cell+adhesion+molecule,+apCAM,+mediates+growth+cone+steering+by+substrate-cytoskeletal+coupling.">The Ig superfamily cell adhesion molecule, apCAM, mediates growth cone steering by substrate-cytoskeletal coupling.</a> J. Cell. Biol. 141, 227-240 (1998).</li><li>Schaefer, A. W., Kabir, N., Forscher, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Filopodia+and+actin+arcs+guide+the+assembly+and+transport+of+two+populations+of+microtubules+with+unique+dynamic+parameters+in+neuronal+growth+cones.">Filopodia and actin arcs guide the assembly and transport of two populations of microtubules with unique dynamic parameters in neuronal growth cones.</a> J. Cell. Biol. 158, 139-152 (2002).</li><li>Suter, D. M., Schaefer, A. W., Forscher, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule+dynamics+are+necessary+for+SRC+family+kinase-dependent+growth+cone+steering.">Microtubule dynamics are necessary for SRC family kinase-dependent growth cone steering.</a> Curr. Biol. 14, 1194-1199 (2004).</li></ol>

Aplysia neuroni delle cellule forniscono una borsa senza siero neuronale sistema di coltura cellulare con pochissime cellule non neuronali. Questi neuroni formano coni di crescita molto grande adatto per affrontare una serie di domande importanti nelle cellule biologiche. I neuroni delle cellule borsa può essere facilmente manipolato e ripreso a temperatura ambiente per diverse ore. Mediante microscopia a fluorescenza Speckle (FSM) si può analizzare quantitativamente i vari parametri di F-actina e polimerizzazione dei microtubuli e la din...

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		<th>Material Name</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>#1 glass coverslips 22x22 mm</td>
<td>Tool</td>
<td>VWR</td>
<td>48366067</td>
<td>&nbsp;</td>
<tr>
<td>#1.5 glass coverslips 22x22 mm</td>
<td>Tool</td>
<td>Corning</td>
<td>2870-22</td>
<td>&nbsp;</td>
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<td>35 mm Petri dishes</td>
<td>Tool</td>
<td>Falcon</td>
<td>353001</td>
<td>&nbsp;</td>
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<td>Filters for medium filtration</td>
<td>Tool</td>
<td>Millipore</td>
<td>SVGV010RS</td>
<td>&nbsp;</td>
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<td>High vacuum grease</td>
<td>Tool</td>
<td>Dow Corning</td>
<td>1597418</td>
<td>&nbsp;</td>
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<td>Osmometer</td>
<td>Tool</td>
<td>Wescor</td>
<td>5520</td>
<td>&nbsp;</td>
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<td>Plastic shims</td>
<td>Tool</td>
<td>Small Parts Inc </td>
<td>SHSP-200</td>
<td>&nbsp;</td>
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<td>Calcium chloride</td>
<td>Reagent</td>
<td>Fisher</td>
<td>C79-500</td>
<td>&nbsp;</td>
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<td>Gentamicin</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>15750-060</td>
<td>&nbsp;</td>
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<td>HEPES</td>
<td>Reagent</td>
<td>Sigma</td>
<td>H4030</td>
<td>&nbsp;</td>
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<td>L15 medium</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>41300-039</td>
<td>&nbsp;</td>
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<td>L-glutamine</td>
<td>Reagent</td>
<td>Sigma</td>
<td>G8540</td>
<td>&nbsp;</td>
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<td>Magnesium chloride</td>
<td>Reagent</td>
<td>Mallinckrodt</td>
<td>5958-04</td>
<td>&nbsp;</td>
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<td>Magnesium sulfate</td>
<td>Reagent</td>
<td>Mallinckrodt</td>
<td>6070-12</td>
<td>&nbsp;</td>
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<td>Poly-L-lysine (70-150 kD) </td>
<td>Reagent</td>
<td>Sigma</td>
<td>P6282</td>
<td>&nbsp;</td>
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<td>Sodium chloride</td>
<td>Reagent</td>
<td>Mallinckrodt </td>
<td>7581</td>
<td>&nbsp;</td>
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<td>Neutral Protease (Dispase)</td>
<td>Reagent</td>
<td>Worthington</td>
<td>LS02111</td>
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neuronal cell cultures from aplysia for high-resolution imaging of growth cones

Coni di crescita neuronali sono strutture altamente mobili sulla punta degli assoni in grado di rilevare segnali di orientamento per l&#39;ambiente e trasducono queste informazioni in movimento direzionale verso la cellula bersaglio appropriato. Per comprendere pienamente come le informazioni di guida viene trasmessa dalla superficie cellulare al connesse reti dinamica del citoscheletro, si ha la necessità di un sistema modello adatto per l&#39;imaging di cellule in vivo di proteine ​​dinamica ad alta risoluzione temporale e spaziale. Tipici coni di crescita vertebrati sono troppo piccoli per analizzare quantitativamente F-actina e la dinamica dei microtubuli. I neuroni dalla lepre mare Aplysia californica sono 5-10 volte più grande di neuroni vertebrati, può facilmente essere conservato a temperatura ambiente e sono cellule molto robusto per micromanipolazione e misure biofisici. I loro coni di crescita sono ben definite regioni citoplasmatiche e ben descritto sistema del citoscheletro. I corpi delle cellule neuronali possono essere microiniettati con una varietà di sonde per studiare la motilità del cono di crescita e di orientamento. Nel presente protocollo si dimostra una procedura per la dissezione del ganglio addominale, la cultura dei neuroni delle cellule borsa e la creazione di una camera di imaging per l&#39;imaging cellulare dal vivo di coni di crescita.

Watch this Scientific Journal Video about Neuronal Cell Cultures from Aplysia for High-Resolution Imaging of Growth Cones at JoVE.com

Neuronal Cell Cultures from Aplysia for High-Resolution Imaging of Growth Cones

Aplysia californica neuroni sviluppare grandi coni di crescita nella cultura che sono eccellenti per imaging ad alta risoluzione della motilità del cono di crescita e di orientamento. Qui, vi presentiamo un protocollo per la dissezione e la placcatura dei neuroni borsa cellule Aplysia così come per la creazione di una camera per l&#39;imaging cellulare dal vivo.

Colture cellulari neuronali da Aplysia per imaging ad alta risoluzione di coni di crescita

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information ...

neuronal-cell-cultures-from-aplysia-for-high-resolution-imaging

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JoVE Journal

Biology

11.3K Views.  Purdue University. Aplysia californica neuroni sviluppare grandi coni di crescita nella cultura che sono eccellenti per imaging ad alta risoluzione della motilità del cono di crescita e di orientamento. Qui, vi presentiamo un protocollo per la dissezione e la placcatura dei neuroni borsa cellule Aplysia così come per la creazione di una camera per l'imaging cellulare dal vivo.

Video: Neuronal Cell Cultures from Aplysia for High-Resolution Imaging of Growth Cones

Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the removal of brains from animals at 70-78 hrs after puparium formation. The isolated brains are shown after brief incubation in papain followed by several washes in serum-free growth medium. The process of mechanical dissociation of each brain in a 5 ul drop of media on a coverslip is illustrated. The axons and dendrites of the post-mitotic neurons are sheered off near the soma during dissociation but the neurons begin to regenerate processes within a few hours of plating. Images show live cultures at 2 days. Neurons continue to elaborate processes during the first week in culture. Specific neuronal populations can be identified in culture using GAL4 lines to drive tissue specific expression of fluorescent markers such as  GFP or RFP. Whole cell recordings have demonstrated the cultured neurons form functional, spontaneously active cholinergic and GABAergic synapses. A short video segment illustrates calcium dynamics in the cultured neurons using Fura-2 as a calcium indicator dye to monitor spontaneous calcium transients and nicotine evoked calcium responses in a dish of cultured neurons. These pupal brain cultures are a useful model system in which genetic and pharmacological tools can be used to identify intrinsic and extrinsic factors that influence formation and function of central synapses.

This video demonstrates the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. Views of live cultures show neurite outgrowth and imaging of calcium levels using Fura-2.

Culture primarie neuronale dal cervello di tardo fase Drosophila Pupae

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the ...

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Biologia

Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos and their dechorionation using bleach are demonstrated. Using a glass pipet attached to a mouth suction tube, we illustrate the removal of all cells from single embryos. The method for dispersing cells from each embyro into a small (5 l) drop of medium on an uncoated glass coverslip is demonstrated. A view through the microscope at 1 hour after plating illustrates the preferred cell density. Most of the cells that survive when grown in defined medium are neuroblasts that divide one or more times in culture before extending neuritic processes by 12-24 hours. A view through the microscope illustrates the level of neurite outgrowth and branching expected in a healthy culture at 2 days in vitro. The cultures are grown in a simple bicarbonate based defined medium, in a 5% CO2 incubator at 22-24&deg;C. Neuritic processes continue to elaborate over the first week in culture and when they make contact with neurites from neighboring cells they often form functional synaptic connections. Neurons in these cultures express voltage-gated sodium, calcium, and potassium channels and are electrically excitable. This culture system is useful for studying molecular genetic and environmental factors that regulate neuronal differentiation, excitability, and synapse formation/function.

This video demonstrates the preparation of primary neuronal cultures from midgastrula stage Drosophila embryos. Views of live cultures show cells 1 hour after plating and differentiated neurons after 2 days of growth in a bicarbonate-based defined medium. The neurons are electrically excitable and form synaptic connections.

Preparazione delle Culture neuronale da Midgastrula fase embrioni di Drosophila

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos ...

Preparing T Cell Growth Factor from Rat Splenocytes

Maintenance of antigen-specific T cell lines or clones in culture requires rounds of antigen-induced activation separated by phases of cell expansion 1,2. Addition of interleukin 2 to the culture media during the expansion phase is necessary to prevent cell death and sufficient to maintain short-term T cell lines but has been shown to increase Th1 polarization 3. Replacement of interleukin 2 by T cell growth factor (TCGF) which contains a mix of cytokines is more effective than interleukin 2 in maintaining long-term T cell lines in vitro 3. Moreover, TCGF can easily be prepared in large amounts in the laboratory and is much cheaper than recombinant interleukin 2. 
Here, we show how to prepare TCGF from rat splenocyte culture supernatants. For this procedure, we harvest spleens from naive Lewis rats euthanized for thymus and blood collection. We prepare single-cell suspensions from the spleens, lyze the red blood cells by osmotic shock, and seed the splenocytes in culture medium. The cells are stimulated with concanavalin A, a mitogen that non-selectively activates all rat T lymphocytes, inducing the production of cytokines. The culture supernantant is collected 48 hours later andexcess concanavalin A is bound to alpha methyl mannoside to prevent it from activating T cell lines to which TCGF will be added. The TCGF is then sterile-filtered, aliquoted, and stored at -20&deg;C.

We describe the preparation of T cell growth factor used for the in vitro expansion of antigen-specific rat T lymphocyte lines.

Preparazione T fattore di crescita cellulare da splenociti Rat

Maintenance of antigen-specific T cell lines or clones in culture requires rounds of antigen-induced activation separated by phases of cell expansion 1,2. ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

La preparazione di colture primarie di cellule emopoietiche dal midollo osseo murino per elettroporazione

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field. Studying locomotor behavior in Drosophila melanogaster relies on the ability to be able to quantify changes in motion during or in response to a given task. For this reason, a high-resolution video tracking system, such as the one we describe in this paper, is a valuable tool for measuring locomotion in real-time. Our protocol involves the use of an initial air pulse to break the flies momentum, followed by a thirty second filming period in a square chamber. A tracking program is then used to calculate the instantaneous speed of each fly within the chamber in 10 msec increments. Analysis software then compiles this data, and outputs a variety of parameters such as average speed, max speed, time spent in motion, acceleration, etc. This protocol will discuss proper feeding and management of flies for behavioral tasks, handling flies without anesthetization or immobilization, setting up a controlled environment, and running the assay from start to finish.

The study of complex locomotor behavior in Drosophila melanogaster is dependent upon the ability to quantify changes in a given fly's movement. This article demonstrates how to do this using a high-resolution tracking system.

Monitoraggio ad alta risoluzione video di locomozione per adulti in Drosophila melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field.  Studying ...

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons. The neurons are large (up to 1 mm in diameter) and identifiable, with distinct sizes, shapes, positions and pigmentations, and the cell bodies are externally exposed in five paired ganglia distributed throughout the body of the animal. These properties have allowed investigators to delineate the circuitry underlying specific behaviors in the animal1. The monosynaptic connection between sensory and motor neurons is a central component of the gill-withdrawal reflex in the animal, a simple defensive reflex in which the animal withdraws its gill in response to tactile stimulation of the siphon. This reflex undergoes forms of non-associative and associative learning, including sensitization, habituation and classical conditioning. Of particular benefit to the study of synaptic plasticity, the sensory-motor synapse can be reconstituted in culture, where well-characterized stimuli elicit forms of plasticity that have direct correlates in the behavior of the animal2,3. Specifically, application of serotonin produces a synaptic strengthening that, depending on the application protocol, lasts for minutes (short-term facilitation), hours (intermediate-term facilitation) or days (long-term facilitation). In contrast, application of the peptide transmitter FMRFamide produces a synaptic weakening or depression that, depending on the application protocol, can last from minutes to days (long-term depression). The large size of the neurons allows for repeated sharp electrode recording of synaptic strength over periods of days together with microinjection of expression vectors, siRNAs and other compounds to target specific signaling cascades and molecules and thereby identify the molecular and cell biological steps that underlie the changes in synaptic efficacy.
An additional advantage of the Aplysia culture system comes from the fact that the neurons demonstrate synapse-specificity in culture4,5. Thus, sensory neurons do not form synapses with themselves (autapses) or with other sensory neurons, nor do they form synapses with non-target identified motor neurons in culture. The varicosities, sites of synaptic contact between sensory and motor neurons, are large enough (2-7 microns in diameter) to allow synapse formation (as well as changes in synaptic morphology) with target motor neurons to be studied at the light microscopic level.
In this video, we demonstrate each step of preparing sensory-motor neuron cultures, including anesthetizing adult and juvenile Aplysia, dissecting their ganglia, protease digestion of the ganglia, removal of the connective tissue by microdissection, identification of both sensory and motor neurons and removal of each cell type by microdissection, plating of the motor neuron, addition of the sensory neuron and manipulation of the sensory neurite to form contact with the cultured motor neuron.

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

Primary cultures of Aplysia sensory-motor neurons provide a model preparation for studying synapse formation and synaptic plasticity in vitro. This video demonstrates the identification and microdissection of sensory and motor neurons from Aplysia ganglia as well as the methods for establishing and maintaining sensory-motor neurons in culture.

Preparazione del Aplysia Senso-motorio colture cellulari neuronali

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons.  The neurons are large (up ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

Colture cellulari primarie da Drosophila Gastrula embrioni

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

Generation of Dispersed Presomitic Mesoderm Cell Cultures for Imaging of the Zebrafish Segmentation Clock in Single Cells

Segmentation is a periodic and sequential morphogenetic process in vertebrates. This rhythmic formation of blocks of tissue called somites along the body axis is evidence of a genetic oscillator patterning the developing embryo. In zebrafish, the intracellular clock driving segmentation is comprised of members of the Her/Hes transcription factor family organized into negative feedback loops. We have recently generated transgenic fluorescent reporter lines for the cyclic gene her1 that recapitulate the spatio-temporal pattern of oscillations in the presomitic mesoderm (PSM). Using these lines, we developed an in vitro culture system that allows real-time analysis of segmentation clock oscillations within single, isolated PSM cells. By removing PSM tissue from transgenic embryos and then dispersing cells from oscillating regions onto glass-bottom dishes, we generated cultures suitable for time-lapse imaging of fluorescence signal from individual clock cells. This approach provides an experimental and conceptual framework for direct manipulation of the segmentation clock with unprecedented single-cell resolution, allowing its cell-autonomous and tissue-level properties to be distinguished and dissected.

Somitogenesis is a rhythmic developmental process that spatially patterns the body axis of vertebrate embryos. Previously, we developed transgenic zebrafish lines that use fluorescent reporters to observe the cyclic genes that drive this process. Here, we culture dispersed cells from these lines and image their oscillations over time in vitro.

Generazione di Dispersi Presomitic mesoderma colture cellulari, per Immagini del Zebrafish segmentazione Orologio in celle singole

Segmentation is a  periodic and sequential morphogenetic process in vertebrates. This rhythmic  formation of blocks of tissue called somites along the ...

Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

An ex vivo technique for imaging mouse airway epithelia for quantitative analysis of motile cilia function important for insight into mucociliary clearance function has been established. Freshly harvested mouse trachea is cut longitudinally through the trachealis muscle and mounted in a shallow walled chamber on a glass-bottomed dish. The trachea sample is positioned along its long axis to take advantage of the trachealis muscle to curl longitudinally. This allows imaging of ciliary motion in the profile view along the entire tracheal length. Videos at 200 frames/sec are obtained using differential interference contrast microscopy and a high speed digital camera to allow quantitative analysis of cilia beat frequency and ciliary waveform. With the addition of fluorescent beads during imaging, cilia generated fluid flow also can be determined. The protocol time spans approximately 30 min, with 5 min for chamber preparation, 5-10 min for sample mounting, and 10-15 min for videomicroscopy.

Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

An easy and reliable technique for visualizing and quantifying airway cilia motility and cilia generated flow using mouse trachea is described. This technique can be modified to determine how a wide range of factors influence cilia motility, including pharmacological agents, genetic factors, environmental exposures, and/or mechanical factors such as mucus load.

Metodo ex vivo per l&#39;imaging ad alta risoluzione di Cilia Motilità in roditori Airway epiteli

An ex vivo technique for imaging mouse airway epithelia for quantitative  analysis of motile cilia function important for insight into mucociliary  ...

Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics1,2. One of the most well-known and widely-utilized +TIPs for analyzing MT dynamics is the End-Binding protein, EB1, which binds all growing MT plus-ends, and thus, is a marker for MT polymerization1. Many studies of EB1 behavior within growth cones have used time-consuming and biased computer-assisted, hand-tracking methods to analyze individual MTs1-3. Our approach is to quantify global parameters of MT dynamics using the software package, plusTipTracker4, following the acquisition of high-resolution, live images of tagged EB1 in cultured embryonic growth cones5. This software is a MATLAB-based, open-source, user-friendly package that combines automated detection, tracking, visualization, and analysis for movies of fluorescently-labeled +TIPs. Here, we present the protocol for using plusTipTracker for the analysis of fluorescently-labeled +TIP comets in cultured Xenopus laevis growth cones. However, this software can also be used to characterize MT dynamics in various cell types6-8.

Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

The MATLAB-based, open source software package, plusTipTracker, can be used to analyze image series of fluorescently-labeled +TIPs to quantify microtubule dynamics.

Utilizzo plusTipTracker software per misurare Microtubulo Dynamics in<em&gt; Xenopus laevis</em&gt; coni di crescita

Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics1,2. One of the most well-known and ...