Questo lavoro è stato sostenuto da NIH concedere NS27501 a DKOD. Supporto aggiuntivo per questo lavoro è stato fornito da una concessione di UC Irvine a sostegno della DKOD attraverso il Programma Professore HHMI.

I preparativi prima del giorno di coltura: <ol><li> Fai la soluzione sterile dissezione. </li><li> Rendere sterile DMEM e mantenere in aliquote da 10 ml a 4 ° C per 2 settimane. </li><li> Rendere sterile DDM2 integratori e congelare in 50 o aliquote di 100 microlitri per 1 mese. </li><li> Fai ConA / laminina. </li><li> Cappotto coprioggetto. Opzionale: Fai CNBM sterile e conservare congelati per un massimo di 4 mesi. </li></ol> Il giorno di coltura I. Preparare soluzione enzimatica (ES) in cappa a flusso laminare <ol><li> Mettere 5 ml di soluzione di dissezione (DS) in una provetta da centrifuga da 15 ml. </li><li> Pesare 0,8 mg di L-cisteina in una Eppendorf 0,6 ml e aggiungere 150 ml di DS. Pipetta su e giù fino cristalli sono andati. </li><li> Aggiungere 150 ml di DS / Cisteina ai 5 ml DS e mescolare bene. </li><li> Aggiungere 50 U (unità) di papaina. </li><li> Aggiungere 7 ml di 0,1 N Na OH e mescolare bene. Soluzione sarà chiara entro 30 min. quando viene attivato. </li><li> Enzima soluzione filtro con filtro da 0,2 millimetri in una siringa sterile tubo a vite microcentrifuga 1,5 ml tappo Papaina: Volete 50 U in 5 ml DS. Ogni lotto è leggermente diverso, così bisogna calcolare quanto: 37,3 mg / ml e 28,3 U / mg 37,3 x 28,3 = 1055,59 U / ml (1000 ml) 50 U = 47,4 ml </li></ol> II. Fai DDM2 dei media Del giorno di coltura: aggiungere i seguenti supplementi per rendere DDM2 A 10 ml di DMEM aggiungere supplementi, poco prima di coltura: <ol><li> 100μl transferrina </li><li> 100μl Putrescine </li><li> 100μl Selenio </li><li> Progesterone 100μl </li><li> L&#39;insulina 50μl </li><li> 10μl 20-hydroxyecdysone </li></ol> Nota: abbastanza DDM2 per tutte le culture pianificata (ogni cervello placcato su un unico vetrino in una teglia da 35 millimetri individuale Petri, 1,5 ms di media / cultura) Fase III-VI può essere a una panchina fatta di laboratorio non sterili e dovrebbe essere completato in 45 minuti o meno. III. Pupe Collection <ol><li> Seleziona 10-15 pupe da più parti di fiala sotto un microscopio da dissezione. </li><li> Pupe posto nel coperchio di un piatto mm secco 35 Petri. </li></ol> Nota: In genere uso il cervello da pupe tra 55-78 ore dopo la formazione puparium (APF). E &#39;leggermente più difficile da sezionare il cervello dalle pupe più giovani dato che le capsule testa tendono a collassare, mentre il livello globale della crescita dei neuriti è leggermente inferiore dal più antiche pupe. Nel Canton-S ceppo tipo selvatico, queste fasi sono riconosciuti da occhi pigmentati che sono marrone chiaro al rossiccio leggermente, con poco pigmento altrove. IV. Decapitazione sotto microscopio da dissezione <ol><li> Mettere due gocce di soluzione sterile dissezione nel coperchio della piastra Petri, vicino a pupe. </li><li> Premere delicatamente una sola pupa con una pinza sottile nella mano sinistra e utilizzare un 27 ago della siringa attaccato ad una siringa da 1 cc nella mano destra per rimuovere la cuticola alla fine anteriore della pupa. </li><li> Spremere pupa leggermente con una pinza e come capo emerge utilizzare i 27 gauge a tagliare il collo. </li><li> Con la punta di un ago o la pinza, il trasferimento testa a goccia di dissezione soluzione. </li><li> Ripetere fino ad avere 10-15 teste in una sola goccia. </li></ol> Rimozione V. del cervello dalla testa sotto microscopio da dissezione <ol><li> In ogni mano mantenere una siringa da 1 cc con un ago da 27 gauge. </li><li> Usare questi per posizionare la testa vola nella goccia di soluzione in modo da sezionare la proboscide è rivolto verso l&#39;alto e la superficie dorsale della testa è a nord. </li><li> Inserire l&#39;ago a sinistra nella proboscide al pin la testa in giù e utilizzare l&#39;ago diritto di presentare una fessura nell&#39;occhio destro che va dal ventrale verso la superficie dorsale. </li><li> Inserire l&#39;ago proprio vicino alla sinistra nella regione della proboscide e quindi utilizzare l&#39;ago a sinistra per premere delicatamente la cuticola sopra l&#39;occhio sinistro, spingendo il cervello verso la fessura nell&#39;occhio destro. Il cervello deve uscire dalla fessura a destra, relativamente intatte, spesso con entrambe le ottiche lobes ancora attaccato e talvolta il tessuto oculare pigmentato pure. </li><li> Spingere il cervello sul retro del l&#39;ago e trasferire a una goccia di soluzione salina sterile dissezione in una nuova sterile, 35 millimetri piatto di Petri. </li><li> Raccogliere 10-15 cervelli per ogni genotipo. </li></ol> VI. Rimozione dei lobi ottici e trattamento enzimatico <ol><li> In ogni mano mantenere una siringa da 1 cc con un ago da 27 gauge e utilizzare questi per raccogliere tutti i cervelli in una piccola area della goccia di soluzione di dissezione </li><li> Utilizzare siringhe, utensili da taglio per rimuovere i lobi ottici da ogni cervello in modo che il tessuto rimanente per la cultura è la regione centrale del cervello. </li><li> Usare un 20 l Pipetman, fissato al 5 microlitri, per trasferire tutti i cervelli di un singolo genotipo di una provetta Eppendorf contenente 1 ml di soluzione di papaina sterile. </li><li> Cervelli incubati in papaina per 10-15 minuti su un insieme dei rotatori a 60 giri. </li><li> Centrifugare a 3000 rpm per 3 minuti. </li></ol> All&#39;interno cappa sterile a flusso laminare - tutte le fasi in cui è esposto il tessuto all&#39;aria dovrebbe essere fatto in cappa a flusso laminare per i passaggi rimanenti. VII. Lavaggio <ol><li> Rimuovere soluzione enzimatica e sostituire con soluzione sterile dissezione. </li><li> Centrifugare a 3000 rpm per 3 minuti. </li><li> Lavare con soluzione sterile dissezione per un totale di 3 volte. </li><li> Rimuovere salina sezionare e sostituire con DDM2. </li><li> Centrifuga e lavare un totale di 2 volte con DDM2. </li><li> Trasferimento di cervelli e mezzi di comunicazione a un piatto sterile 35 millimetri Petri. </li></ol> VIII. Triturazione e placcatura in microscopio da dissezione <ol><li> Mettere 5 ul di DDM2 al centro di un vetrino coprioggetto. </li><li> Trasferire 1 cervello per questo calo di DDM2 con una punta di giallo. </li><li> Sotto il microscopio, utilizzare aghi calibro 27 a dadi il cervello di in piccoli pezzi (dovrebbe prendere &lt;1 minuto). </li><li> Sotto la guida visiva, rompere la punta di una pipetta di vetro che è stato tirato a punta fine in modo che i pezzi più grandi possono essere risucchiati delicatamente la punta della pipetta ed espulso di nuovo nel mezzo. Utilizziamo tubi di aspirazione bocca che sono forniti di serie con 100 microlitri di vetro ematocrito capillare. Nota: Fare attenzione a non avere le bolle nei media e sarà necessario determinare empiricamente la pipetta dimensione migliore da usare. Buoni consentono di dissociare il cervello con alcune singole cellule e ancora alcuni cespugli di grandi dimensioni, in circa 30 secondi / cerebrale. Quando si guarda a queste a maggior potenza, circa il 10-20% dei neuroni devono ancora alcuni processi rimanenti e ci saranno ancora alcuni grumi di grandi dimensioni. Se si dissociano troppo, allora tutte le cellule saranno rotondi e avranno subito danni così tanto che non sopravviverà. Questa fase richiede pratica e deve essere fatto in fretta perché c&#39;è una grande superficie-volume e il rapporto osmolarità e pH della goccia di DDM2 possono cambiare rapidamente. Tessuto deve essere posto in CO 2 incubatore nel più breve tempo possibile. </li><li> Scrivi sul coperchio capsula di Petri: &quot;genotipo, data e ora&quot;. </li><li> Mettere il piatto 35 millimetri petri in una capsula di Petri mm 100 (con kimwipe bagnato) e lasciare riposare in CO 2 incubatore per 30 min. </li><li> Inondare il piatto 35 mm con 1,5 ml di DDM2. </li><li> Mantenere le culture in incubatrice al 5% CO 2 incubatore (23 ° C). Nota: se non si ha accesso ad un raffreddamento CO 2 incubatore, utilizzare uno standard di mammifero tessuto cultura incubatore e sia messo in una stanza fresca e calore a 23 ° C, o mettere in laboratorio, spegnere temp, e l&#39;uso ghiaccio in un cassetto nella parte inferiore del incubatore per regolare temperatura ambiente intorno. </li></ol> IX. Le cellule di alimentazione <ol><li> In occasione della Giornata 1 (giorno successivo), aggiungere 0,5 ml di CNBM/B27 (Media Condizionata) per rendere 03:01 DDM2: CNBM/B27. </li><li> Il 5 ° giorno sostituire 1 ml di media per DDM2 03:01: CNBM/B27. </li><li> Conservare le cellule di alimentazione con 3:1 DDM2: CNBM/B27 ogni 4-5 giorni. Nota: Le culture possono essere coltivate senza i media condizionata non neuronali, ma i neuroni sembrano un po &#39;più fragili e sono più difficili per l&#39;uso negli esperimenti di elettrofisiologia. </li></ol>

<ol>
	<li>Banker, G., Goslin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culturing Nerve Cells. , (1991).</li><li>Rohrbough, J., O'Dowd, D. K., Baines, R. A., Broadie, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+bases+of+behavioral+plasticity:+Establishing+and+Modifying+synaptic+circuits+in+the+Drosophila+Genetic+System.">Cellular bases of behavioral plasticity: Establishing and Modifying synaptic circuits in the Drosophila Genetic System.</a> J. Neurobiol. 54, 254-271 (2003).</li><li>O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltage-gated+currents+and+firing+properties+of+embryonic+Drosophila+neurons+grown+in+a+chemically+defined+medium.">Voltage-gated currents and firing properties of embryonic Drosophila neurons grown in a chemically defined medium.</a> J. Neurobiol. 27, 113-126 (1995).</li><li>Lee, D., O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+excitatory+synaptic+transmission+mediated+by+nAChR+in+cultured+Drosophila+neurons.">Fast excitatory synaptic transmission mediated by nAChR in cultured Drosophila neurons.</a> J. Neurosci. 19, 5311-5321 (1999).</li><li>Su, H., O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+synaptic+currents+in+Drosophila+mushroom+body+Kenyon+cells+are+mediated+by+alpha-bungarotoxin-sensitive+nAChRs+and+picrotoxin-sensitive+GABA+receptors.">Fast synaptic currents in Drosophila mushroom body Kenyon cells are mediated by alpha-bungarotoxin-sensitive nAChRs and picrotoxin-sensitive GABA receptors.</a> J. Neurosci. 23, 9246-9253 (2003).</li><li>Jiang, S. A., Campusano, J. M., Su, H., O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+mushroom+body+Kenyon+cells+generate+spontaneous+calcium+transients+mediated+by+PLTX-sensitive+calcium+channels.">Drosophila mushroom body Kenyon cells generate spontaneous calcium transients mediated by PLTX-sensitive calcium channels.</a> J. Neurophysiol. 94, 491-500 (2005).</li><li>Campusano, J. M., Su, H., Jiang, S. A., Sicaeros, B., O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=nAChR-mediated+calcium+responses+and+plasticity+in+Drosophila+Kenyon+cells.">nAChR-mediated calcium responses and plasticity in Drosophila Kenyon cells.</a> Develop Neurobiol. 67, 1520-1532 (2007).</li><li>Oh, H. -. W., Campusano, J. M., Hilgenberg, L. G. W., Sun, X., Smith, M. A., O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructural+analysis+of+chemical+synapses+and+gap+junctions+between+Drosophila+brain+neurons+in+culture.">Ultrastructural analysis of chemical synapses and gap junctions between Drosophila brain neurons in culture.</a> Develop Neurobiol. , (2007).</li></ol>

I neuroni raccolte dal cervello di roditori embrionale / postnatale possono essere coltivate in coltura cellulare primaria dove si estendono neuriti e la forma funzionale connessioni sinaptiche. Metodi per la preparazione di queste culture sono ben stabiliti e gli studi in colture neuronali roditori hanno svolto un ruolo fondamentale per identificare geni e fattori ambientali coinvolti nella regolazione della formazione di sinapsi e la funzione (Banker e Goslin, 1991). Mentre i neuroni degli insetti da una varietà di specie possono essere ...

Vetrini coprioggetto rivestimento: Mettere coprioggetto autoclave in un piatto da 60 millimetri di Petri. Pipettare 5 ul di Con A / mix laminina nella parte centrale di ogni coprioggetto. Luogo in 37 C incubatore per 2 ore. Sciacquare coprioggetto 3x con 100 ul di acqua autoclavata ciascuno. Usa vuoto attaccati ad una pipetta Pasteur sterile. Durante l&#39;ultimo risciacquo, sollevare il vetrino con una pinza ed asciugare entrambi i lati. Trasferire il vetrino di un piatto 35 millimetri Petri. Conservare a temperatura ambiente per un massimo di un mese.

primary neuronal cultures from the brains of late stage drosophila pupae

In questo video, dimostriamo la preparazione di colture primarie neuronali dal cervello di fine tappa Drosophila pupe. La procedura inizia con la rimozione di cervelli di animali a 70-78 ore dopo la formazione puparium. I cervelli isolati sono mostrati dopo incubazione breve papaina seguito da numerosi lavaggi in mezzo privo di siero di crescita. Il processo di dissociazione meccanica di ogni cervello in un calo del 5 ul di media su un vetrino è illustrato. Gli assoni e dendriti del post-mitotico neuroni sono sheered in prossimità del soma durante dissociazione, ma i neuroni cominciano a rigenerare i processi in poche ore di placcatura. Immagini mostrano fermenti vivi a 2 giorni. I neuroni continuano a elaborare processi durante la prima settimana della cultura. Specifiche popolazioni di neuroni possono essere identificati in coltura utilizzando GAL4 linee per guidare l&#39;espressione di marcatori specifici del tessuto fluorescente come GFP o RFP. Registrazioni di cellule intere hanno dimostrato l&#39;neuroni coltivati ​​forma funzionale, sinapsi colinergica attiva spontaneamente e GABAergici. Un segmento breve video che illustra le dinamiche del calcio nei neuroni in coltura con Fura-2 come un colorante indicatore di calcio di monitorare transienti di calcio spontanea e nicotina risposte di calcio evocate in un piatto di neuroni in coltura. Queste culture cervello pupa sono un sistema modello utile in cui gli strumenti genetica e farmacologica può essere utilizzato per identificare i fattori intrinseci ed estrinseci che influenzano la formazione e la funzione delle sinapsi centrale.

Watch this Scientific Journal Video about Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae at JoVE.com

Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

Questo video dimostra la preparazione di colture primarie neuronali dal cervello di fine tappa Drosophila pupe. Visto di culture live show crescita dei neuriti e l&#39;imaging di livelli di calcio con 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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15.0K Views.  University of California, Irvine (UCI). Questo video dimostra la preparazione di colture primarie neuronali dal cervello di fine tappa Drosophila pupe. Visto di culture live show crescita dei neuriti e l'imaging di livelli di calcio con Fura-2.

Video: Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

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 ...

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

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 into directional movement towards the appropriate target cell. To fully understand how guidance information is transmitted from the cell surface to the underlying dynamic cytoskeletal networks, one needs a model system suitable for live cell imaging of protein dynamics at high temporal and spatial resolution. Typical vertebrate growth cones are too small to quantitatively analyze F-actin and microtubule dynamics. Neurons from the sea hare Aplysia californica are 5-10 times larger than vertebrate neurons, can easily be kept at room temperature and are very robust cells for micromanipulation and biophysical measurements. Their growth cones have very defined cytoplasmic regions and a well-described cytoskeletal system. The neuronal cell bodies can be microinjected with a variety of probes for studying growth cone motility and guidance. In the present protocol we demonstrate a procedure for dissection of the abdominal ganglion, culture of bag cell neurons and setting up an imaging chamber for live cell imaging of growth cones.

Aplysia californica neurons develop large growth cones in culture that are excellent for high-resolution imaging of growth cone motility and guidance. Here, we present a protocol for dissection and plating of Aplysia bag cell neurons as well as for setting up a chamber for live cell imaging.

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 ...

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation from systemic input from elsewhere in the brain. In our lab, we use these neurons to assess dopamine release kinetics using carbon fiber amperometry, as well as expression levels of dopamine related genes and proteins using quantitative PCR and immunocytochemistry. In this video, we show you how we generate these cultures from rodent neonates.
The process involves several steps, including the plating of cortical glial astrocytes, the conditioning of neuronal cell culture media by the glial substrate, the dissection of the midbrain in neonates, the digestion, extraction and plating of dopamine neurons and the addition of neurotrophic factors to ensure cell survival.
The applications suitable for such a preparation include electrophysiology, immunocytochemistry, quantitative PCR, video microscopy (i.e., of real-time vesicular fusion with the plasma membrane), cell viability assays and other toxicological screens.

Primary dissociated midbrain dopamine cell cultures allow for the study of presynaptic characteristics of dopamine neurons. They can be used to monitor real-time dopamine release kinetics and protein/mRNA levels of regulators of dopamine exocytosis. Here, we show you how to generate these cultures from rodent neonates.

Primaria dissociato mesencefalo colture cellulari dopamina da roditori neonati

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation ...

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 ...

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 ...

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three preparations of the brain and visual system that cover the range from the developing eye disc-brain complex in the developing pupae to individual eye and brain dissection from adult flies. All protocols are optimized for the live culture of the preparations. However, we also present the conditions for fixed tissue immunohistochemistry where applicable. Finally, we show live imaging conditions for these preparations using conventional and resonant 4D confocal live imaging in a perfusion chamber. Together, these protocols provide a basis for live imaging on different time scales ranging from functional intracellular assays on the scale of minutes to developmental or degenerative processes on the scale of many hours.

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

This protocol describes three Drosophila preparations: 1) adult brain dissection, 2) adult retina dissection and 3) developing eye disc- brain complexes dissection. Emphasis is laid on special preparation techniques and conditions for live imaging, although all preparations can be used for fixed tissue immunohistochemistry.

Preparazione di Sviluppo e degli adulti Drosophila Brains e retina per l&#39;imaging dal vivo

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three ...

Establishing Primary Adult Fibroblast Cultures From Rodents

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized. Primary cells are preferred in studies of cell cycle control, apoptosis, and DNA repair, as cancer cells carry mutations in genes involved in these processes. Primary cells cannot be cultured indefinitely due to the onset of replicative senescence or aneuploidization. Hence, new cultures need to be established regularly. The procedure for isolation of rodent embryonic fibroblasts is well established, but isolating adult fibroblast cultures often presents a challenge. Adult rodent fibroblasts isolated from mouse models of human disease may be a preferred control when comparing them to fibroblasts from human patients. Furthermore, adult fibroblasts are the only available material when working with wild rodents where pregnant females cannot be easily obtained. Here we provide a protocol for isolation and culture of adult fibroblasts from rodent skin and lungs. We used this procedure successfully to isolate fibroblasts from over twenty rodent species from laboratory mice and rats to wild rodents such as beaver, porcupine, and squirrel.

This article describes a protocol for isolation and maintenance of primary fibroblast cultures from skin and lung tissue of wild rodents.

Stabilire colture primarie di fibroblasti adulti dai roditori

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized.  Primary cells are preferred ...

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 ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&amp;#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

Naïve Adult Stem Cells isolamento da colture primarie di fibroblasti umani

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

The Utility of Stage-specific Mid-to-late Drosophila Follicle Isolation

Drosophila oogenesis or follicle development has been widely used to advance the understanding of complex developmental and cell biologic processes. This methods paper describes how to isolate mid-to-late stage follicles (Stage 10B-14) and utilize them to provide new insights into the molecular and morphologic events occurring during tight windows of developmental time. Isolated follicles can be used for a variety of experimental techniques, including in vitro development assays, live imaging, mRNA expression analysis and western blot analysis of proteins. Follicles at Stage 10B (S10B) or later will complete development in culture; this allows one to combine genetic or pharmacologic perturbations with in vitro development to define the effects of such manipulations on the processes occurring during specific periods of development. Additionally, because these follicles develop in culture, they are ideally suited for live imaging studies, which often reveal new mechanisms that mediate morphological events. Isolated follicles can also be used for molecular analyses. For example, changes in gene expression that result from genetic perturbations can be defined for specific developmental windows. Additionally, protein level, stability, and/or posttranslational modification state during a particular stage of follicle development can be examined through western blot analyses. Thus, stage-specific isolation of Drosophila follicles provides a rich source of information into widely conserved processes of development and morphogenesis.

The Utility of Stage-specific Mid-to-late Drosophila Follicle Isolation

Stage-specific isolation of mid-to-late Drosophila follicles is useful for a variety of purposes. Such follicles develop in culture, which allows for genetic and/or pharmacologic manipulations to be coupled with in vitro development assays and live imaging. Additionally, follicles can be used for molecular studies, such as isolating mRNA and protein.

L&#39;utilità della fase-specifici medio-tardiva<em&gt; Drosophila</em&gt; Follicolo isolamento

Drosophila oogenesis or follicle development has been widely used to advance the understanding of complex developmental and cell biologic processes. This ...