Questo studio è stato finanziato in parte da fondi di start-up della Texas A &amp; M University

Preparazione prima della dissezione Strumenti chirurgici: sterilizzare tutti forbici e pinze da dissezione autoclave Coprioggetto pulizia: <ol><li> Immergere tutti coprioggetto (15 mm di diametro per 24-pozzetti) in 1L bicchiere erba nel 33% di HCl per almeno 24 ore </li><li> Lavare con acqua corrente per 10 minuti con scuotere occasionali per rimuovere tutti i residui di HCl </li><li> Risciacquare con acqua Millipore </li><li> Scaricare l&#39;acqua fuori </li><li> Mettere a bagno in coprioggetto 95-98% di etanolo </li><li> Coprioggetto trasferimento in un tessuto pulito in un piatto piano e aria secca i coprioggetti </li><li> Trasferimento coprioggetto ad un bicchiere di vetro, coprite con un foglio di alluminio e autoclave Nota: Vetrini coprioggetto possono essere memorizzati in questa fase </li><li> Luogo coprioggetto individuali in piastre di coltura. Pronto per la verniciatura </li></ol> Coprioggetto rivestimento: <ol><li> Prelevare 100 x magazzino di poli-D-lisina (PDL, 10 mg / ml in 0,5% di BSA in PBS, immagazzinato come aliquote a -20 ° C) con PBS e filtro-sterilizzare (0,22 micron) </li><li> Coprioggetto cappotto con 1 soluzione x PDL (~ 0,15 ml / cm 2) per 2 ore a 37 ° C in un incubatore </li><li> Rimuovere la soluzione PDL e lavare 3 volte con sterili DDH 2 O e coprioggetto asciugare completamente </li></ol> Nota: Cappotto abbastanza lamelle / piastre per ogni dissezione. PDL rivestite coprioggetto può essere conservato per un paio di settimane a 4 ° C. Mezzi di comunicazione e soluzioni Medio dissezione (DM): Utilizzare soluzione equilibrata sale sterili ghiacciata di Hank (w / o Ca 2 +, Mg 2 +) (HBSS, Invitrogen 14175) integrata con 10 mM HEPES (Invitrogen 15.630). Terreni di coltura di aggregazione: Il mezzo DMEM/NBB27 contiene DMEM / Neurobasal (1:1 vol: vol), 2% B27, 1 x Sato, 0,5 mM di piruvato di sodio, 0,75 mm GlutaMAX, 60μg/ml N-acetilcisteina, 5 mcg / ml insulina, 10nm d-biotina e 1% di penicillina / streptomicina. Per fare 100 ml di terreno di coltura aggregato, miscelare 50 ml di DMEM (w / o piruvato / glutammina, Invitrogen 11960), 50 ml di mezzo Neurobasal (Invitrogen 211.034), 375 microlitri GlutaMax (100 x, Invitrogen 35.050), 5,5 mg di sodio piruvato (Sigma P2256), 2 ml di B27 (Invitrogen 17.504), 6,3 mg di N-Acetilcisteina (Sigma A8199), 1 ml di brodo Sato (100 x magazzino, vedi sotto), 25 l di d-Biotina magazzino (4000 x magazzino, 40 mM in PBS memorizzati come aliquote a -20 ° C. Sigma B4639), 100 ml di insulina (1000 x stock, 5 mg / ml di HCl 0.01N memorizzati come aliquote a -20 ° C, Sigma 16634) e 1 ml di penicillina / streptomicina (100 x stock, Invitrogen 15.140), filtro-sterilizzare e conservare a 4 ° C. Sato 100 x soluzione di riserva: Per 40 ml dello stock: mescolare 40 Neurobasal ml con 400 mg di apo-transferrina (Sigma T2252), 400 mg di BSA (Sigma A9647), 10 ml di progesterone (25 mg / ml di etanolo, memorizzati come aliquote a -20 ° C. Sigma P8783), 64 mg putrescina (Sigma P7505), e 40 l di sodio selenite (30 mM in PBS, immagazzinato come aliquote a -20 ° C, Sigma S5261). Sterilizzare filtro della soluzione madre Sato, e conservare come aliquote a -20 ° C. Neurone medio placcatura (PM): Medio Neurobasal, 2% B27, 2mM Glutammina (stock 100x, aliquote conservati a -20 ° C, Invitrogen 25.030), a 25 micron acido glutammico (100 stock, aliquote conservati a -20 ° C) e 1% penicillina / streptomicina. Neuron terreno di coltura (CM): Medio Neurobasal, 2% B27, 2mM Glutammina (stock 100x, aliquote conservati a -20 ° C) e 1% di penicillina / streptomicina. 5-FDU magazzino: magazzino 100x, 1mm 5-fluoro-2&#39;-deossiuridina (Sigma F0503) and1mM uridina (Sigma U3003) in un mezzo Neurobasal. Sterilizzato per filtrazione e memorizzati come aliquote a -20 ° C. Papaina soluzione di digestione (fare ogni volta prima dissezione): <ol><li> Sciogliere 3,2 mg L-cisteina (Sigma C-7352) in 4ml DM </li><li> Regolare il pH a circa 7,4 con NaOH 1N (test su strisce reattive pH) e posto in bagno d&#39;acqua a 37 ° C Nota: eseguire il passo successivo a destra prima di digestione dei tessuti (vedi sotto). </li><li> Aggiungi papaina ad una concentrazione finale di 20 unità / ml </li><li> Filtro sterilizzare (0,22 millimetri), e posto in bagno d&#39;acqua a 37 ° C </li></ol> Soluzione di tripsina inibitore (fare ogni volta prima dissezione): <ol><li> Sciogliere 0,2 g di inibitori della tripsina (Sigma T7295) a 20ml di DM </li><li> Controllare il pH e aggiustare il pH a ~ 7.4 con 1 N NaOH </li><li> Filtro sterilizzare, e posto in bagno d&#39;acqua a 37 ° C </li></ol> La dissezione del cervello Setup: <ul><li> Pinze e forbici sterilizzate </li><li> Etanolo al 70% </li><li> Fazzoletto di carta pulito e DIAPer pad sulla panchina </li><li> Petri dieci centimetri di ghiaccio-freddo DM per utero e feto sulla panchina </li><li> Piattaforma di ghiaccio al microscopio dissezione </li><li> 2-3 piatti con DM 6 centimetri sul ghiaccio </li><li> PM caldo in un bagno d&#39;acqua a 37 ° C </li></ul><ol><li> Euthanize una donna incinta ratto Sprague-Dawley (E16) secondo una procedura approvata dal Animal Care istituzionali e Us Committee (IACUC) </li><li> Posare il ratto sulla rampa di pannolino e l&#39;alcool spay sulla regione addominale </li><li> Utilizzare una pinzetta per tenere la cute addominale e un paio di forbici per fare una incisione forma a V taglio solo la pelle </li><li> Allargate la pelle e utilizzare un altro pinzette sterilizzate e forbici per tagliare lo strato muscolare </li><li> Estrarre la catena delle sacche dell&#39;embrione e trasferirli in un piatto di 10 centimetri contenente ghiacciata DM </li><li> Utilizzando un paio di forbici microdissezione, rimuovere con attenzione ogni embrione dal sacco e il cervello da ogni embrione, e il luogo liberato cervello in un piatto pulito, con DM 10 centimetri su una piattaforma di ghiaccio </li><li> In una cappa laminare e al microscopio dissezione, rimuovere mesencefalo / rombencefalo sezioni, e utilizzare una pinza sottile per rimuovere meningi, e trasferire le cortecce pulito / ippocampo di un nuovo piatto con DM 6 centimetri su un impacco di ghiaccio Nota: in questa fase, il tessuto è pronto per la digestione enzimatica. </li><li> Fai la papaina e la soluzione di inibitori della tripsina e filtro sterilizzare </li><li> Rimuovere l&#39;eccesso di DM da cortecce / dell&#39;ippocampo </li><li> Aggiungere 4 ml della soluzione preparata digestione papaina </li><li> Trasferire tutto il contenuto in una provetta da 50 ml e posizionare il tubo Falcon in un bagno d&#39;acqua a 37 ° C per esattamente 5 minuti </li><li> Rimuovere soluzione papaina con pipetta </li><li> Aggiungere 5 ml di soluzione di inibitori della tripsina, agitare il tubo, e posto in un bagno di acqua a 37 ° C per 2-3 minuti </li><li> Rimuovere soluzione inibitore della tripsina </li><li> Ripetere il punto 13 e 14 tre volte </li><li> Aggiungere 20ml PM caldo </li><li> Triturare fino a quando tutti i grumi sono scomparsi (~ 20-30 volte) </li><li> Centrifugare a 100xg per 7min </li><li> Risospendere il pellet in 10ml PM e lavare due volte per centrifugazione </li><li> Risospendere il pellet in 10ml PM per le culture dei neuroni regolari o in NBB27/DMEM per aggregare culture </li><li> Passaggio delle cellule attraverso un setaccio cellula 70μm </li><li> Contare le cellule vive </li><li> Procedere alle culture aggregato come descritto di seguito o la targa delle celle per le culture dei neuroni regolare Nota: A questo punto, altamente arricchito culture neurone può essere ottenuto placcatura cellule dissociate sul PDL-rivestimento piastre a densità di 200-640 cellule / mm 2 a seconda dello scopo degli esperimenti. Dopo che le cellule hanno attaccato (~ 2-4h), sostituire medio con PM caldo. Se coltura per lunghi periodi di tempo, il giorno 4, sostituire la metà dei media con CM caldo. Per altamente purificato culture neuronali, inibitore della 5-mitotico FDU (concentrazione finale 10 micron) possono essere aggiunte in DIV2 per inibire la non proliferazione delle cellule neuronali (cioè durante DIV2-3). Dopo il recupero di CM per 2 giorni, le cellule sono impulsi nuovamente trattati con 5-FDU per altri 2 giorni (DIV 6-7) seguito da un cambiamento di terreno completo. In seguito, sostituire la metà della media con fresco, CM calda ogni 3-4 giorni. I neuroni sono vitali per un massimo di 4 settimane. </li></ol> Aggregati preparazione e placcatura <ol><li> Sospendere le cellule dissociate in media NBB27/DMEM contenente 1x Sato, 10ng/ml CNTF e 10μM forskolina ad una densità di 2x10 6 cellule / ml </li><li> Trasferimento 2 ml di sospensione cellulare in ciascun pozzetto di una non rivestito da 6 pozzetti </li><li> Cultura per 3 notti. Ogni giorno, risospendere delicatamente le cellule una volta con un Pipetman P1000 Nota: Le cellule iniziano a formare aggregati (Figura 1). </li><li> Il giorno prima della placcatura aggregato, cappotto piastra / coprioggetto con Matrigel: <ul><li> Diluire la Matrigel (fattore di crescita ridotta, BD Biosciences 354230) 1:20 con DMEM freddo sul ghiaccio Nota: aliquote Matrigel devono essere preparati per ogni protocollo del produttore. Tutte le punte, tubi Eppendorf e soluzioni per rendere aliquote dello stock dovrebbe essere fredda per evitare la polimerizzazione Matrigel. Aliquote di Matrigel sono conservati a -80 ° C e scongelato a 4 ° C. </li><li> Cappotto in precedenza PDL rivestite coprioggetto con 300μl/well della soluzione diluita Matrigel durante la notte a 37 ° C incubatore </li><li> Lavare con PBS caldo, quindi lavare con piastra calda sterile DDH 2 O e lasciare le piastre in incubatrice. </li></ul></li><li> 3 giorni dopo la formazione di aggregati, risospendere delicatamente le cellule di nuovo e setaccio la sospensione attraverso una maglia 200μm in un tubo da 50 ml Falcon. Lasciare aggregati di depositarsi sul fondo della provetta per gravità (~ 3-5min) </li><li> Rimuovere con cura surnatante </li><li> Aggiungere mezzo, risospendere delicatamente gli aggregati e lasciarli depositare di nuovo. Ripetere questa operazione più volte per rimuovere le cellule morte, le singole cellule e detriti. Usa microscopio per verificare se il supernatante contengono ancora non aggregata cellule e detritis </li><li> Risospendere delicatamente gli aggregati nel medio NBB27/DMEM stesso (Figura 1) e numero di aggregati </li><li> Regolare la densità di aggregati per circa 25-30 aggreggats/50ul </li><li> Aliquote di trasferimento (500-1000μl) della sospensione aggregato di 2ml provette Eppendorf per la placcatura Nota: poiché aggregati tendono a stabilirsi sul fondo dei tubi, è fondamentale per rendere più tubi della sospensione aggregato per la placcatura. Risospendere delicatamente gli aggregati spesso prima di loro semina su vetrini rivestiti o piastre di coltura per assicurare numero simile di aggregati placcato su ogni vetrino. </li><li> Estrarre la piastra di coltura contenente Matrigel rivestite coprioggetti dai incubatrice. Rimuovere la soluzione da ogni pozzetto, lavare i coprioggetti con caldo PBS e DDH 2 O, e poi brevemente a secco. Essi sono ora pronti per la semina aggregato </li><li> Invertire una provetta contenente sospensione aggregata per consentire loro di essere distribuiti in modo uniforme prima della semina. Carico 50μl di sospensione aggregato al centro di ogni coprioggetto, e gentilmente messo la piastra posteriore per incubatrice senza alcun disturbo per consentire ulteriori aggregazioni di collegare in modo uniforme alla coprioggetto Nota: La densità degli aggregati è fondamentale. Se aggregati sono seminati troppo vicini gli uni agli altri o se la densità di semina è alta, tendono a fondersi man mano che crescono. </li><li> Aggiungi DMEM/NBB27medium aggiuntivi (500 microlitri) in ogni pozzetto 4-6 ore più tardi o la mattina presto </li><li> Per mantenere la cultura aggregato, la metà cambiare mezzo ogni 3-4 giorni. Quando il cambiamento medio, fare attenzione a non disturbare gli aggregati e questo è fatto da dolci aggiungendo medio lungo la parete del pozzo. Nota: Poche ore dopo aggregati attaccato alla piastra, crescita dei neuriti dagli aggregati possono essere osservati. Assoni crescere rapidamente nelle prime due settimane e formare connessioni abbondante tra gli aggregati (Figura 2). Progenitori gliali migrano radialmente fuori degli aggregati e si differenziano nel tempo in astrociti e oligodendrociti maturi. </li></ol><img src="/files/ftp_upload/1304/1304fig1.jpg" alt="figura 1" /> Figura 1. Contrasto di fase immagini di cellule che formano aggregati in giorni diversi e aggregati dopo il lavaggio. <img src="/files/ftp_upload/1304/1304fig2.jpg" alt="figura 2" /> Figura 2. Contrasto di fase le immagini di una cultura aggregato 2 e 12 giorni dopo seminato su Matrigel rivestite coprioggetto.

<ol>
	<li>Matthieu, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myelination+in+rat+brain+aggregating+cell+cultures.">Myelination in rat brain aggregating cell cultures.</a> Neuroscience. 3, 565-56 (1978).</li><li>Chen, Y. i. n. g. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+rat+and+mouse+oligodendrocyte+precursor+cells.">Isolation and culture of rat and mouse oligodendrocyte precursor cells.</a> Nat. Protocols. 2, 1044-10 (2007).</li><li>Watkins, T. A., Emery, B., Mulinyawe, S., Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+stages+of+myelination+regulated+by+gamma-secretase+and+astrocytes+in+a+rapidly+myelinating+CNS+coculture+system.">Distinct stages of myelination regulated by gamma-secretase and astrocytes in a rapidly myelinating CNS coculture system.</a> Neuron. 60, 555-55 (2008).</li><li>Svenningsen, A. F., Shan, W. e. i. -. S. o. n. g., Colman, D. a. v. i. d. R., Pedraza, L. i. l. i. a. n. a. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+method+for+culturing+embryonic+neuron-glial+cell+cocultures.">Rapid method for culturing embryonic neuron-glial cell cocultures.</a> Journal of Neuroscience Research. 72, 565-56 (2003).</li></ol>

Primi studi riportavano la formazione di sinapsi e mielina maturo in rotazione-mediata culture senza aggregato galleggianti 1. L&#39;aggregato SNC sistema di coltura qui descritto combina senza siero crescita delle cellule progenitrici multipotenti aggregati in tre dimensioni con la comodità di culture tradizionali in 2D per facilitare le analisi dello sviluppo delle cellule, la migrazione e la differenziazione in vitro. Il sistema può essere modificato e utilizzato per precursore neuronali ricerca...

preparation of rat brain aggregate cultures for neuron and glia development studies

Un sistema in vitro che ricapitola lo sviluppo e la differenziazione dei progenitori in neuroni maturi e glia nel sistema nervoso centrale (SNC) potrebbe fornire una piattaforma potente per i neuroscienziati di indagare AXO-gliali interazioni, le proprietà e la differenziazione di cellule progenitrici multipotenti, e la progressione della oligodendrogliale cellule lignaggio a livello cellulare e molecolare. Descriviamo qui un aggregato SNC sistema di coltura da forebrains ratto embrionale, che può essere mantenuta in un mezzo privo di siero fino a 3-4 settimane ed è utilizzato nel nostro laboratorio come modello per lo studio dei neuroni-glia l&#39;interazione e la mielinizzazione del SNC. Questo video verrà illustrato come isolare e coltivare queste aggregato SNC culture E16 cervello di ratto. Inoltre, dalla dissezione del cervello stesso, altamente arricchito regolare culture neuronali dissociate possono essere ottenuti facilmente e utilizzati per vari studi sui neuroni del SNC o utilizzato per co-culture con altre cellule.

Watch this Scientific Journal Video about Preparation of Rat Brain Aggregate Cultures for Neuron and Glia Development Studies at JoVE.com

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

Un protocollo per un sistema embrionale del cervello di ratto cultura globale è descritto. Progenitori multipotenti negli aggregati in grado di sviluppare e differenziarsi in neuroni, astrociti e oligodendrociti.

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) ...

preparation-rat-brain-aggregate-cultures-for-neuron-glia-development

Research

JoVE Journal

Biology

12.3K Views.  Texas A&M University (TAMU). Un protocollo per un sistema embrionale del cervello di ratto cultura globale è descritto. Progenitori multipotenti negli aggregati in grado di sviluppare e differenziarsi in neuroni, astrociti e oligodendrociti.

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

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 ...

Ricerca

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 ...

ALS - Motor Neuron Disease: Mechanism and Development of New Therapies

Jeffrey D. Rothstein speaks about the pathology and mechanisms underlying amyotrophic lateral sclerosis or ALS, advances in ALS research, and current strategies towards the development of therapies.

SLA - Motor Neuron Disease: meccanismo e sviluppo di nuove terapie

Fabrication of a Microfluidic Device for the Compartmentalization of Neuron Soma and Axons

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for culturing neurons. Previously, a silicon wafer was patterned with the design for the neuron microfluidic device using SU-8 and photolithography to create a master mold, or what we simply refer to as a "master". Next, we pour the silicon polymer PDMS on top of the master which is then cured by heating the PDMS to 80&deg;C for 1 hour. The PDMS forms a negative mold of the device. The PDMS is then carefully cut and lifted away from the master. Holes are punched where the reservoirs will be and the excess PDMS trimmed away from the device. Nitrogen is used to blow away any excess debris from the device. At this point the devices are now ready for use and can either bonded to corning No. 1 cover glass with a plasma sterilizer/cleaner or can be reversibly bound to the cover glass by simply placing the device on top of the cover glass. The reversible bonding of the device to glass is covered in a separate video and requires first that the device be sterilized either with 70% ethanol or by autoclaving. Plasma treating sterilizes the devices so no further treatment is necessary. It is, however, important, when plasma-treating the devices, to add liquid to the devices within 10 minutes of the plasma treatment while the surfaces are still hydrophilic. Waiting longer than 10 minutes to add liquid to the device makes it difficult for the liquid to enter the device. The neuron devices are typically plasma-bound to cover glass and 0.5 mg/ml poly-L-lysine (PLL) in pH 8.5 borate buffer is immediately added to the device. After a minimum of 3 hours incubating with PLL, the devices are washed with dH2O water a minimum of 3 times with at least 15 minutes between each wash. Next, the water is removed and fresh media is added to the device. At this point the device is ready for use. It is important to remember at this point to never remove all the media from the device. Always leave media in the main channel.

In this video we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to farbricate a microfluidic device for culturing neurons.

Fabbricazione di un dispositivo a microfluidi per la compartimentazione dei Neuron Soma e assoni

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for ...

Preparation of Dissociated Mouse Cortical Neuron Cultures

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures can be used for a variety of applications including immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging, protein and/or RNA isolation. These cultures also provide a platform to study the neuronal development of transgenic animals that carry a late embryonic or postnatal lethal gene mutation. The procedure is relatively straight forward, requires some experience in tissue culture technique and should not take longer than two to three hours if you are properly prepared. Careful separation of the cortical rind from the thalamo-cortical fiber tract will reduce the number of unwanted non-neuronal cells. To increase yields of neuronal cells triturate the pieces of the cortical tissue gently after the enzyme incubation step. This is imperative as it prevents unnecessary injury to cells and premature neuronal cell death. Since these cultures are maintained in the absence of glia feeder cells, they also offer an added advantage of growing cultures enriched in neurons.

This video shows a procedure for generating neuronal cultures from late embryo and early postnatal mouse cortex. These cultures can be used for immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging and provide a platform to study the neuronal development of transgenic animals that carry a postnatal lethal gene mutation.

Preparazione del dissociato Culture Neuron mouse corticale

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures ...

Preparation of 2-dGuo-Treated Thymus Organ Cultures

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play a key role in the development of a functionally competent T-cell pool. However, the complexity of T-cell development in the thymus in vivo can limit analysis of individual cellular components and particular stages of development. In vitro culture systems provide a readily accessible means to study multiple complex cellular processes. Thymus organ culture systems represent a widely used approach to study intrathymic development of T-cells under defined conditions in vitro. Here we describe a system in which mouse embryonic thymus lobes can be depleted of endogenous haemopoeitic elements by prior organ culture in 2-deoxyguanosine, a compound that is selectively toxic to haemopoeitic cells. As well as providing a readily accessible source of thymic stromal cells to investigate the role of thymic microenvironments in the development and selection of T-cells, this technique also underpins further experimental approaches that include the reconstitution of alymphoid thymus lobes in vitro with defined haemopoietic elements, the transplantation of alymphoid thymuses into recipient mice, and the formation of reaggregate thymus organ cultures. (This article is based on work first reported Methods in Molecular Biology 2007, Vol. 380 pages 185-196).

This video demonstrates the dissection and removal of the fetal thymus as well the preparation of ex vivo cultures of 2-dGuo-treated thymus.

La preparazione di 2-dGuo-trattati Organ Culture Timo

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play ...

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 ...

A Multi-compartment CNS Neuron-glia Co-culture Microfluidic Platform

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to six independent experiments in parallel for higher-throughput. We developed a new fabrication method to create microfluidic devices having both micro and macro scale structures within the same device through a single soft-lithography process, enabling mass fabrication with good repeatability.
The multi-compartment microfluidic co-culture platform is composed of one soma compartment for neurons and six axon/glia compartments for oligodendrocytes (OLs). The soma compartment and axon/glia compartments are connected by arrays of axon-guiding microchannels that function as physical barriers to confine neuronal soma in the soma compartment, while allowing axons to grow into axon/glia compartments. OLs loaded into axon/glia compartments can interact only with axons but not with neuronal soma or dendrites, enabling localized axon-glia interaction studies. The microchannels also enabled fluidic isolation between compartments, allowing six independent experiments to be conducted on a single device for higher throughput.
Soft-lithography using poly(dimethylsiloxane) (PDMS) is a commonly used technique in biomedical microdevices. Reservoirs on these devices are commonly defined by manual punching. Although simple, poor alignment and time consuming nature of the process makes this process not suitable when large numbers of reservoirs have to be repeatedly created. The newly developed method did not require manual punching of reservoirs, overcoming such limitations. First, seven reservoirs (depth: 3.5 mm) were made on a poly(methyl methacrylate) (PMMA) block using a micro-milling machine. Then, arrays of ridge microstructures, fabricated on a glass substrate, were hot-embossed against the PMMA block to define microchannels that connect the soma and axon/glia compartments. This process resulted in macro-scale reservoirs (3.5 mm) and micro-scale channels (2.5 &mu;m) to coincide within a single PMMA master. A PDMS replica that served as a mold master was obtained using soft-lithography and the final PDMS device was replicated from this master.
Primary neurons from E16-18 rats were loaded to the soma compartment and cultured for two weeks. After one week of cell culture, axons crossed microchannels and formed axonal only network layer inside axon/glia compartments. Axons grew uniformly throughout six axon/glia compartments and OLs from P1-2 rats were added to axon/glia compartments at 14 days in vitro for co-culture.

We developed a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research. The platform is capable of conducting up to six independent experiments in parallel and was fabricated using a newly developed macro/micro hybrid fabrication method.

Un multi-vano SNC Neuron-glia Co-cultura Microfluidic piattaforma

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to ...

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

Fusaria are filamentous fungi able to produce different toxins. Fusarium mycotoxins such as deoxynivalenol, nivalenol, T2, zearelenone, fusaric acid, moniliformin, etc... have adverse effects on both human and animal health and some are considered as pathogenicity factors. Proteomic studies showed to be effective for deciphering toxin production mechanisms (Taylor et al., 2008) as well as for identifying potential pathogenic factors (Paper et al., 2007, Houterman et al., 2007) in Fusaria. It becomes therefore fundamental to establish reliable methods for comparing between proteomic studies in order to rely on true differences found in protein expression among experiments, strains and laboratories. The procedure that will be described should contribute to an increased level of standardization of proteomic procedures by two ways. The filmed protocol is used to increase the level of details that can be described precisely. Moreover, the availability of standardized procedures to process biological replicates should guarantee a higher robustness of data, taking into account also the human factor within the technical reproducibility of the extraction procedure.
The protocol described requires 16 days for its completion: fourteen days for cultures and two days for protein extraction (figure 1). 
Briefly, Fusarium strains are grown on solid media for 4 days; they are then manually fragmented and transferred into a modified toxin inducing media (Jiao et al., 2008) for 10 days. Mycelium is collected by filtration through a Miracloth layer. Grinding is performed in a cold chamber. Different operators performed extraction replicates (n=3) in order to take into account the bias due to technical variations (figure 2). Extraction was based on a SDS/DTT buffer as described in Taylor et al. (2008) with slight modifications. Total protein extraction required a precipitation process of the proteins using Aceton/TCA/DTT buffer overnight and Acetone /DTT washing (figure 3a,3b). Proteins were finally resolubilized in the protein-labelling buffer and quantified. Results of the extraction were visualized on a 1D gel (Figure 4, SDS-PAGE), before proceeding to 2D gels (IEF/SDS-PAGE). The same procedure can be applied for proteomic analyses on other growing media and other filamentous fungi (Miles et al., 2007).

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

Protein extraction for proteomic analyses in fungal species requires high levels of standardization to be accomplished according with the minimum information about a proteomic experiment (MIAPE) guidelines. We present a video-protocol that includes a procedure for minimizing experimental bias during toxin induction and protein extraction from Fusarium spp.

Tossina induzione ed estrazione di proteine ​​da Fusarium Spp. Culture per studi di proteomica

Fusaria are filamentous fungi able to produce different toxins. Fusarium mycotoxins such as deoxynivalenol, nivalenol, T2, zearelenone, fusaric acid, ...

Preparation of Rat Tail Tendons for Biomechanical and Mechanobiological Studies

Rat tail tendons (RTTs) are a common biological model used in experimental in vitro studies in the fields of tendon physiology and tendinopathy. Working with those tissues is challenging because they are very fragile, and until now there was no rigorously detailed protocol for their isolation.
Faced with these challenges, we have developed methods and instruments to facilitate manipulation of RTTs and control tissue viability, sterility and integrity. This article describes the experimental procedures used to prepare RTTs for biomechanical and mechanobiological studies. Our work is divided into four main steps: extraction, cross-sectional area measurement, rinsing and loading into the bioreactor chamber.
At each step, all procedures, materials and manipulations are presented in detail so that they can be easily reproduced. Moreover, the specific instruments developed are presented: a manipulation plate used to segregate RTTs, an optic micrometer to position the tissue during the cross-sectional area measurement and an anchoring system to attach the RTTs onto a bioreactor.
Finally, we describe the results obtained after multiple tests to validate our methods. The viability, sterility and integrity evaluations demonstrate that our procedures are sufficiently rigorous for manipulations of fragile tissues such as rat tail tendons.

This article describes the experimental procedures used to prepare rat tail tendons for biomechanical and mechanobiological studies. Several features of the main steps in preparation are demonstrated, beginning with extraction, cross-sectional area measurement, rinsing and loading into the bioreactor chamber.

Preparazione dei tendini coda di topo per studi di biomeccanica e Mechanobiological

Rat tail tendons (RTTs) are a common biological model used in experimental in vitro studies in the fields of tendon physiology and tendinopathy. Working ...