I preparativi prima del giorno di coltura: <ul><li> Preparare soluzione sterile dissezione (DS). </li><li> Preparare NBM/B27 (Mediom Neurobasal con supplementi di B27). </li><li> Autoclave DDH 2 O e sterilizzare coversilps vetro, se necessario. </li><li> Cappotto tessuto piatti cultura o coprioggetto di vetro con poli-D-lisina. </li></ul> Poli-D-lisina Coating: Preparare il giorno prima della coltura in condizioni di sterilità. <ul><li> Aliquota del disgelo PDL 10X e posto sul ghiaccio. </li><li> Aggiungere 9 ml sterile ultra-filtrata acqua a 1 ml di PDL e mescolare bene (1X). </li><li> Superfici cappotto con 1X Pdl notte a temperatura ambiente come segue: <ul><li> vetro coprioggetti (Bellco gatto di vetro, Inc. # 1943-00012.): 75 microlitri / coprioggetto O </li><li> 24 pozzetti cultura: 300 ml / pozzetto O </li><li> 35 millimetri piatto cultura: 1ml/dish </li></ul></li><li> Risciacquare con acqua sterile 5X (usare pipette Pasteur sterili per aspirare liquidi). </li><li> Eliminare l&#39;acqua in superficie fino a quando è completamente asciutto. </li></ul> Coltura procedura (fatto in cappa a flusso laminare in condizioni sterili) <ol><li> Tagliare blocco di agar e la colla sul blocco sostegno della Vibraslicer microtomo (Campden Instruments Ltd.) con colla super. </li><li> Quando si utilizza tardo fase embrionale (E17-18) feti di topo, diga euthanise, togliere utero e feto singolo libero dal sacco embrionale. Feti posto in piastra di Petri sterile e procedere come descritto di seguito. </li><li> Decapitare feto mouse o cucciolo (seguire le linee guida approvate dalla vostra cura degli animali e del Comitato Istituzionale Usa). </li><li> Togliere la pelle e il cranio e il cervello posto sul disco Whatman carta da filtro in una capsula di Petri riempite con 60 millimetri DS freddo. </li><li> Tagliare il cervelletto con una lama di rasoio sterile. </li><li> Pick up cervello con una spatola e scarico liquidi in eccesso su carta da filtro, poi il trasferimento cervello per bloccare il supporto Vibraslicer e il cervello colla al posto (lato caudale e parte ventrale fronte agar). </li><li> Riempire da camera con DS freddo. Impostare il selettore della velocità a 8-9. </li><li> Tagliare sezioni 200-400 micron, a partire dal bulbo olfattivo. Una volta che la lama del Vibraslicer penetri nella corteccia, iniziare il taglio 600μm sezioni coronali. Un cervello E17-18 topo produce tipicamente 3-4 fette utilizzabile, mentre un P0 cervello di topo cede 4-5 fette utilizzabile. </li><li> Trasferimento fettine di cervello di piatto da 35 mm di Petri etichettati DS 2 usando fine inverso di un-inserito 10 ml di vetro pipetta Pasteur. </li><li> Sezionare fuori corteccia e rimuovere le meningi con aghi di vetro tirato da tubi capillari. </li><li> Scorze tagliate corticale in piccoli pezzi, 0,5-1 mm di lunghezza. </li><li> ES Filtrare 0,2 micron filtro attaccato ad una siringa da 5 cc in un piatto 35 millimetri di Petri. </li><li> Trasferire i pezzi di tessuto corticale capsula di Petri contenente ES filtrata. </li><li> Incubare a 37 ° C per 30 min. </li></ol> Nel frattempo: <ol><li> Pulire cappuccio, Vibraslicer e strumenti di dissezione. </li><li> Aggiungere 5 ml di caldo NBM/B27 in un piatto 60 millimetri di Petri. </li></ol> Dopo 30 min. incubazione: <ol><li> Trasferimento di tessuto a 10 ml DS. Lasciare riposare per 1 minuto. </li><li> Trasferimento di tessuto al primo turbinii tubo Hi e lasciare riposare per 2-3 minuti. </li><li> Trasferimento a Hi secondo. Ripetere come sopra. </li><li> Trasferimento al primo Li. Ripetere come sopra. </li><li> Trasferimento alla seconda Li. Ripetere come sopra. </li><li> Cessione a terzi Li. Ripetere come sopra. </li><li> Trasferimento di tessuto al piatto 60 mm con i media. </li></ol> Sotto il microscopio dissezione: <ol><li> Eliminare i detriti dal tessuto e triturare ogni pezzo con delicatezza passando attraverso una pipetta di vetro tirato (minor utilizzo diametri) per allentare il tessuto. </li><li> Trasferimento sospensione cellulare a 15 ml tubo contenente il resto della NBM + B27 (per un totale di 13 ml per una piastra ben 24 o 11 ml per 5 - 35. Piatti millimetri Mescolare delicatamente e attendere grandi pezzi di tessuto per risolvere). </li><li> Trasferimento di sospensione cellulare (0,5 ml / pozzetto di una piastra ben 24 o 2 ml / 35 piatto cultura mm). </li><li> Quando si utilizza coprioggetto di vetro, aggiungere 80 microlitri di sospensione cellulare per coprioggetto e lasciare 1 ora nel tessuto culturale incubatore per le cellule di aderire prima di allagare la camera con più NBM/B27 media. </li><li> Mantenere le culture a 37 ° C e 5% di CO 2. </li></ol> Giorno successivo 24 pozzetti: Feed ciascun pozzetto con 0,5 ml di cellule non neuronali condizionata NBM/B27 medio (cNBM/B27; Protocollo per la preparazione di cNBM/B27 media appare sotto) Piatti 35 mm: Sostituire 0,5 ml con cNBM/B27 media. Mantenere la cultura sostituendo 0,5 ml di media con cNBM/B27 fresca ogni 2-3 giorni. Sebbene i media cultura non promuove la proliferazione delle cellule gliali, le culture possono essere trattate con FDU 5 micron (5-Fluoro-2&#39;-deossiuridina, Sigma F0503) al giorno 3-5 in cultura per ridurre ulteriormente il numero di cellule gliali, se necessario. SET<html> -UP per la dissezione e la Cultura Sterilizzare gli strumenti dissezione nel 70% EtOH o autoclave loro: <ul><li> Forbici (aziende medie e piccole) Spatole (aziende medie e piccole) </li><li> Pinzetta (aziende medie e piccole) Razor blade </li><li> Lama per il bagno Buffer vibraslicer </li><li> Piccolo cuneo di metallo per il supporto della lama per il cervello </li></ul> Altri materiali: <ul><li> Piastre di Petri (60 mm) </li><li> Piastre di Petri (35 mm) </li><li> Filtro di carta </li><li> Pipetta di vetro da 10 ml </li><li> Pipette sterili monouso, 5,10 e 25 ml </li><li> Siringa filtro, 0,2 micron </li><li> Siringa da 5 cc </li><li> Provette per centrifuga, 15 ml </li><li> Sterile di vetro pipette Pasteur </li><li> PDL piatti cultura rivestiti o coprioggetto di vetro </li></ul> Etichetta sei provette da 15 ml e seguire la preparazione: Tubo # 1: soluzione enzimatica: Aggiungere 50 U di papaina (Worthington LS 03126) a 5 DS ml contenente: <ul><li> 100μl di L-cisteina (0,8 mg) </li><li> 7 microlitri 0.1N NaOH </li><li> 50 microlitri APV (5mm) </li></ul> Lasciare la soluzione a temperatura ambiente per cancellare. Si noti che appariranno soluzione enzimatica &#39;torbida&#39; in un primo momento e ha bisogno di pulire prima dell&#39;uso. Tubo # 2 e # 3: inibitore Max: 3 ml DS + 300 ml BSA / Ti + 30 microlitri APV (5mm). Mescolare delicatamente per evitare le bolle, poi dividere in due aliquote da 1,5 ml. Tubo # 4 - # 6: inibitore Basso: 8 ml DS + 80 microlitri BSA / Ti + 80 microlitri APV (5mm). Mescolare delicatamente per evitare le bolle, si dividono in tre aliquote da 2,6 ml. Tubi posto numerato # 2 al # 6 su ghiaccio. Lascia tubo # 1 (soluzione enzimatica) a temperatura ambiente. SOLUZIONI E aliquote <ul><li> Soluzione A (Buffered Saline) Sigma Formula wt 500 ml [conc.] </li><li> Cloruro di sodio NaCl S-9625 58,45 80.0g 137mm </li><li> Cloruro di potassio KCl P-4504 74,56 4,0 G 5.4mm </li><li> Fosfato di sodio bibasico anidro Na 2 HPO 4 S-0876 142,0 0.24g 0,17 mm </li><li> Potassio fosfato monobasico anidro KH 2 PO 4 P-5379 136,09 0,22 mm 0,3 g </li></ul> Pesare tutti gli ingredienti e mescolare fino a sciogliere con 400 ml di acqua ultra filtrata. Portare il volume finale a 500 ml. Mettere in una bottiglia pulita e autoclave. Conservare a 4 ° C e l&#39;etichetta &quot;Soluzione DS A&quot;. <ul><li> Soluzione B (Hepes) Sigma Formula wt 250 ml [conc.] </li><li> Hepes Hepes H-3375 283,3 20.97g 9,9 mm </li></ul> Aggiungi ultra-filtrata l&#39;acqua fino a 200 ml. Mescolare fino a scioglimento e portare il volume finale a 250 ml. Mettere in una bottiglia pulita e autoclave. Conservare a 4 ° C e l&#39;etichetta &quot;DS Soluzione B&quot;. <ul><li> Soluzione di lavoro da 500 ml [conc.] </li><li> Ultra 400 ml di acqua filtrata </li><li> Soluzione A 25 ml </li><li> Soluzione B 14 ml </li><li> D (+)-glucosio Sigma G-8270 3,0 g 33.3mM </li><li> Sigma saccarosio S-0389 7,5 g 43,8 mm </li></ul> Regolare il pH a 7,4 con NaOH 1N. Portare il volume finale a 500 ml con acqua ultra filtrata. Decantare in una bottiglia di vetro pulita e autoclave. Conservare a 4 ° C. Etichetta &quot;Soluzione dissezione&quot;. Poli-D-lisina Preparazione: <ol><li> Preparare la soluzione 10x stock di poli-D-lisina (PDL; Sigma P-7280) in sterili H 2 O a 1mg/ml. </li><li> Fai aliquote 1,0 ml. Questa soluzione può essere conservata a -20 ° C per un massimo di 3 mesi. </li></ol> MEDIA <ol><li> Aggiungere 10 ml di B-27 Supplemento (Gibco 17504-044) per flacone da 500 ml NBM (Medium Neurobasal, Gibco 21103-049). </li><li> Fai 40 aliquote da ml e conservare a 4 ° C. </li></ol> APV <ol><li> Aggiungere 10 ml di H 2 O sterile per flaconcino da 10 mg APV (2-Amino-5-phosphonopentanoic acido, Sigma A-5282) e mescolare accuratamente. </li><li> Preparare aliquote di 180 microlitri e conservare a -20 ° C. </li></ol> BSA / Ti <ol><li> Sciogliere 1 g di BSA (albumina bovina. Sigma A-7030) e 1 g di tripsina inibitore (Sigma T-9253) in 10 ml DS. </li><li> Regolare il pH a 7,4 con NaOH 1N. </li><li> Sterilizzare per filtrazione attraverso filtro da 0,2 micron siringa. </li><li> Dividere in aliquote da 400 microlitri e conservare a -20 ° C. </li></ol> L-cisteina In una provetta Eppendorf sciogliere 0,6 mg L-cisteina (Sigma C-7755) in 200 microlitri DS. AGAR (4%) <ol><li> Sciogliere 4 g di Agar Bacto (Difco 0140-01) in 100 ml di acqua sterile. Conservare a 4 ° C fino al momento della cultura. </li><li> Agar microonde per fondere e riempire un piatto di 35 millimetri di Petri. Lasciate riposare per raffreddare e polimerizzare. </li></ol> Papaina (Worthington LS 03126) e_title &quot;&gt; non neuronali CULTURE IN BOTTIGLIA CULTURA NUNC RIVESTIMENTO BOTTIGLIA CULTURA (4 bottiglie Nunc) <ol><li> Aggiungere 9 ml di acqua sterile a ciascuno dei 3 tubi del PDL 10X per rendere PDL 1X. </li><li> Trasferire 30 ml 1X PDL nella prima bottiglia. </li><li> Spostare leggermente fino a quando tutte le superfici di crescita sono coperti. Lasciate riposare per 1 minuto. </li><li> Trasferimento 1x PDL a seconda bottiglia. Lasciate riposare per 1 minuto. </li><li> Ripetere con le bottiglie terzo e quarto. </li><li> Risciacquare tutte le quattro bottiglie 2X con 150 ml sterile H 2 O. </li></ol> PREPARARE soluzione enzimatica (ES): <ol><li> In un peso 0,6 ml in provetta da centrifuga 0,8 mg L-cisteina (Sigma C7755), aggiungere DS 150ml e vortice fino a quando i cristalli si sciolgono. </li><li> Trasferire 5 ml DS in una provetta da 15 ml, poi aggiungere 150ml L-cisteina. </li><li> Papaina aggiungere 50 unità. (Worthington LS 03126) </li><li> Aggiungi 7ml 0.1N NaOH. </li></ol> PREPARARE MEM (GIBCO # 11090-081) <ol><li> Togliere 65 ml dalla bottiglia da 500 ml MEM. Risparmia il 10 ml per preparare glucosio. </li><li> Aggiungere 5 ml Pen / Strep (Gibco # 15070-063). </li><li> Aggiungere 10 ml di glucosio 1M (Sigma G-8270) (1,8 g / 10 ml di MEM) </li><li> Aggiungere 50 ml di siero fetale bovino (Gibco # 16140-071) o siero bovino di vitello (Omega aC-04). </li><li> Mescolare bene, aliquota e conservare a 4 ° C. </li></ol> NEUROBASAL MEDIUM/B-27 SUPPLEMENTO (NBM/B27) NBM, 500 ml (Gibco # 21103-049), B-27 (50X), 10 ml (Gibco # 17504-044) <ol><li> Aggiungere l&#39;intero contenuto di B-27 vial alla bottiglia NBM e mescolare. </li><li> Aliquota e conservare a 4 ° C. </li></ol> DISSEZIONE del tessuto cerebrale <ol><li> Secco dissezione strumenti dal 70% EtOH prima della dissezione. </li><li> Trasferire 10 ml DS in ogni tubo di 3-15 ml. </li><li> Seleziona cuccioli mouse (P0 - P3), avrete bisogno di tre cervelli per 4 bottiglie. </li><li> Aggiungi DS piatto freddo a 35 mm di Petri. </li><li> Decapitare il mouse cucciolo e sezionare i cervelli. </li><li> Cervello posto nella capsula di Petri contenente DS freddo. </li><li> Tritare tessuto in pezzi, abbastanza piccolo da passare attraverso una pipetta di vetro. </li></ol> ENZIMA DISSOCIAZIONE <ol><li> Rimuovere il DS più possibile il piatto con una pipetta Pasteur. </li><li> ES filtrare attraverso una siringa da 0,2 micron filtro e 5 cc. </li><li> Luogo ES nel piatto con il tessuto. </li><li> Incubare il tessuto a 37 ° C incubatore (CO 2 anni gratis) per 30 minuti. </li></ol> LAVAGGIO DEL TESSUTO <ol><li> Tessuto trasferimento con pipetta di vetro per il primo tubo DS, avendo cura di avere come ES meno possibile. </li><li> Centrifugare per 30 sec a 1500 giri al minuto. </li><li> Trasferimento di tessuti e ripetere la procedura altre due volte. </li></ol> TRITURAZIONE <ol><li> Mettere 38 ml di MEM / FBS in un tubo da 50 ml per centrifuga </li><li> Aggiungere 3 ml di MEM / FBS a un piatto da 35 mm e il tessuto di trasferire a questa capsula di Petri. </li><li> Dissociare il tessuto con delicatezza triturazione attraverso un 1000μl punta della pipetta eppendorf. </li><li> Trasferimento sospensione cellulare alla provetta contenente 38ml di MEM / FBS e mescolare bene. </li></ol> PLATING <ol><li> Stare le bottiglie cultura in posizione verticale per ottenere la stessa quantità di sospensione cellulare e dei media in ogni bottiglia. </li><li> Mettere 90 ml di MEM / FBS in ogni bottiglia cultura. </li><li> Aggiungere 10 ml di sospensione cellulare in ogni bottiglia. </li><li> Etichetta: non neuronali, iniziali e la data. Incubare a 37 ° C e 5% di CO 2. </li></ol> ALIMENTAZIONE <ol><li> Il giorno 3 o 4, le cellule di alimentazione con caldo MEM / FBS (decantare tutti i media e sostituirlo con 100 ml di MEM / FBS). Culture possono prendere 7-10 giorni per diventare confluenti. </li><li> Il giorno di 7-10 cambiare il supporto per NBM/B27. </li><li> Il giorno dopo, raccogliere i media. Filtro con 0.22μm </li><li> Fai aliquote di 40 ml e mangimi cultura con MEM / FBS. </li><li> Prossimi 2 o 3 giorni, cambio dei media per NBM/B27 </li><li> Il giorno dopo, raccogliere e ripetere la procedura. </li><li> Continuare a raccogliere i media per ca. 2 settimane. </li></ol>

The authors have nothing to disclose.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>5-Fluoro-2'-deoxyuridine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>F0503</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Chloride</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S9625</td>
<td>&nbsp;</td>
<tr>
<td>Vibraslicer</td>
<td>&nbsp;</td>
<td>Campden Instruments Ltd.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Potassim Chloride</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P4504</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Phosphate Dibasic</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S0876</td>
<td>&nbsp;</td>
<tr>
<td>Potassium Phosphate Monobasic</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P5379</td>
<td>&nbsp;</td>
<tr>
<td>Hepes</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H3375</td>
<td>&nbsp;</td>
<tr>
<td>D (+)-Glucose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G8270</td>
<td>&nbsp;</td>
<tr>
<td>Sucrose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S0389</td>
<td>&nbsp;</td>
<tr>
<td>Poly-D-Lysine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P7280</td>
<td>&nbsp;</td>
<tr>
<td>B27 Supplement</td>
<td>&nbsp;</td>
<td>Invitrogen (Gibco)</td>
<td>17504-044</td>
<td>&nbsp;</td>
<tr>
<td>Neurobasal Media (NBM)</td>
<td>&nbsp;</td>
<td>Invirtogen (Gibco)</td>
<td>21103-049</td>
<td>&nbsp;</td>
<tr>
<td>2-Amino-5-phosphonopentanoic acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A5282</td>
<td>&nbsp;</td>
<tr>
<td>Bovine Albumin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A7030</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin Inhibitor</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T9253</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Hydroxide, 1N solution</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>SS266-1</td>
<td>&nbsp;</td>
<tr>
<td>L-Cysteine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C7755</td>
<td>&nbsp;</td>
<tr>
<td>Bacto Agar</td>
<td>&nbsp;</td>
<td>Difco</td>
<td>0140-01</td>
<td>&nbsp;</td>
<tr>
<td>Papain</td>
<td>&nbsp;</td>
<td>Worthington </td>
<td>LS 03126</td>
<td>&nbsp;</td>
<tr>
<td>Sterile 0.2 &micro;m Syringe Filter</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>DDA02025S0</td>
<td>&nbsp;</td>
<tr>
<td>Glass Coverslips, No1, &#216;12mm</td>
<td>&nbsp;</td>
<td>Bellco Glass Inc.</td>
<td>1943-00012</td>
<td>&nbsp;</td>
<tr>
<td>Minimum Essential Media (MEM)</td>
<td>&nbsp;</td>
<td>Invitrogen (Gibco)</td>
<td>11090-081</td>
<td>with Earle's salts
without L-glutamine, needed for growing non-neuronal cultures</td>
</tr>
<tr>
<td>Penicillin/Streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen (Gibco)</td>
<td>15070-063</td>
<td>for non-neuronal culture</td>
</tr>
<tr>
<td>Fetal Bovine Serum</td>
<td>&nbsp;</td>
<td>Invitrogen (Gibco)</td>
<td>16140-071</td>
<td>needed for non-neuronal cultures</td>
</tr>
<tr>
<td>Nunclon "Triple Flask" Tissue Culture Bottle</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>12-565-25</td>
<td>Use for non-neuronal cultures. these flasks are very convenient when producing "conditioned Neurobasal Media/B27", but any other tissue culture flasks or dishes can be used instead.</td>
</tr>
<tr>
<td>Glass bottom "Imaging dishes"</td>
<td>&nbsp;</td>
<td>MatTek Corp.</td>
<td>P35G-1.5-10-C</td>
<td>Glass bottomed, &#216;35mm culture dishes ideal for Calcium or Sodium Imaging when using an inverted imaging setup. But expensive!</td>
</tr>		</tbody>
		</table>
		</div>

preparation of dissociated mouse cortical neuron cultures

Questo video vi guiderà attraverso il processo per la generazione di neuroni corticali culture dall&#39;embrione fine e l&#39;inizio del cervello di topo postnatale. Queste culture può essere utilizzato per una varietà di applicazioni, tra cui immunocitochimica, biochimica, elettrofisiologia, imaging di calcio e sodio, proteine ​​e / o RNA isolamento. Queste culture anche fornire una piattaforma per studiare lo sviluppo neuronale di animali transgenici che portano una mutazione in ritardo embrionale o postnatale gene letale. La procedura è relativamente semplice, richiede una certa esperienza nelle tecniche di coltura e non dovrebbe richiedere più di due o tre ore se si è adeguatamente preparati. Un&#39;attenta separazione della crosta corticale dal talamo-corticale del tratto di fibra ridurrà il numero degli indesiderati cellule non neuronali. Per aumentare la resa delle cellule neuronali triturare i pezzi del tessuto corticale delicatamente dopo la fase di incubazione dell&#39;enzima. Questo è indispensabile in quanto previene inutili danni alle cellule e la morte prematura delle cellule neuronali. Dato che queste colture sono mantenute in assenza di cellule di alimentazione glia, offrono anche un vantaggio aggiunto di culture crescente arricchito nei neuroni.

Watch this Scientific Journal Video about Preparation of Dissociated Mouse Cortical Neuron Cultures at JoVE.com

Preparation of Dissociated Mouse Cortical Neuron Cultures

Questo video mostra una procedura per la generazione di culture neuronali dall&#39;embrione fine e l&#39;inizio della corteccia del mouse postnatale. Queste culture possono essere utilizzati per immunocitochimica, biochimica, elettrofisiologia, calcio e sodio di imaging e di fornire una piattaforma per studiare lo sviluppo neuronale di animali transgenici che portano una mutazione genetica postnatale letale.

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-dissociated-mouse-cortical-neuron-cultures

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Biology

43.2K Views.  University of California, Irvine (UCI). Questo video mostra una procedura per la generazione di culture neuronali dall'embrione fine e l'inizio della corteccia del mouse postnatale. Queste culture possono essere utilizzati per immunocitochimica, biochimica, elettrofisiologia, calcio e sodio di imaging e di fornire una piattaforma per studiare lo sviluppo neuronale di animali transgenici che portano una mutazione genetica postnatale letale.

Video: Preparation of Dissociated Mouse Cortical Neuron Cultures

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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Biologia

Mouse Epidermal Neural Crest Stem Cell (EPI-NCSC) Cultures

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the physiological property to generate a wide array of differentiated cell types, including neurons, nerve supporting cells, smooth muscle cells, bone/cartilage cells and melanocytes. EPI-NCSC are easily accessible in the hairy skin and can be isolated as a highly pure population of stem cells. This video provides a detailed protocol for preparing mouse EPI-NCSC cultures from whisker follicles. The whisker pad of an adult mouse is removed, and whisker follicles dissected. The follicles are then cut longitudinally and subsequently transversely above and below the bulge region. The bulge is removed from the collagen capsule and placed in a culture plate. EPI-NCSC start to emigrate from the bulge explants 3 to 4 days later.

Mouse Epidermal Neural Crest Stem Cell EPI-NCSC Cultures

Here we show our method to isolate mouse epidermal neural crest stem cells (EPI-NCSC). Technique involves micro-dissecting whisker follicles, isolating the bulge and placeing it into tissue culture. EPI-NCSC start to emigrate from bulge explants onto the substratum within 3 - 4 days.

Topo Epidermal cresta neurale delle cellule staminali (EPI-NCSC) Culture

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the ...

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 ...

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 and Maintenance of Dorsal Root Ganglia Neurons in Compartmented Cultures

Neurons extend axonal processes that are far removed from the cell body to innervate target tissues, where target-derived growth factors are required for neuronal survival and function. Neurotrophins are specifically required to maintain the survival and differentiation of innervating sensory neurons but the question of how these target-derived neurotrophins communicate to the cell body of innervating neurons has been an area of active research for over 30 years. The most commonly accepted model of how neurotrophin signals reach the cell body proposes that signaling endosomes carry this signal retrogradely along the axon. In order to study retrograde transport, a culture system was originally devised by Robert Campenot, in which cell bodies are isolated from their axons. The technique of preparing these compartmented chambers for culturing sensory neurons recapitulates the selective stimulation of neuron terminals that occurs in vivo following release of target-derived neurotrophins. Retrograde signaling events that require long-range microtubule dependent retrograde transport have important implications for the treatment of neurodegenerative disorders.

Here we describe the technique of preparing and maintaining compartmented chambers for culturing sensory neurons of the dorsal root ganglia.

Preparazione e manutenzione dei neuroni dei gangli dorsali in Culture Compartmented

Neurons extend axonal processes that are far removed from the cell body to innervate target tissues, where target-derived growth factors are required for ...

Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons 

The cerebellar cortex is a well described structure that provides unique opportunities for studying neuronal properties and development1,2. Of the cerebellar neuronal types (granule cells, Purkinje cells and inhibitory interneurons), granule neurons are by far the most numerous and are the most abundant type of neurons in the mammalian brain. In rodents, cerebellar granule neurons are generated during the first two post-natal weeks from progenitor cells in the outermost layer of the cerebellar cortex, the external granule layer (EGL). The protocol presented here describes techniques to enrich and culture granule neurons and their progenitor cells from post-natal mouse cerebellum. We will describe procedures to obtain cultures of increasing purity3,4, which can be used to study the differentiation of proliferating progenitor cells into granule neurons5,6. Once the progenitor cells differentiate, the cultures also provide a homogenous population of granule neurons for experimental manipulation and characterization of phenomena such as synaptogenesis, glutamate receptor function7, interaction with other purified cerebellar cells8,9 or cell death7.

Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons

Here we present a method to isolate and culture cerebellar granule neuron progenitor cells and cerebellar granule neurons from postnatal mouse.

Isolamento e la coltura di cellule post-Natal Neuron cerebellare granuli mouse progenitore e Neuroni

The cerebellar cortex is a well described structure that provides unique opportunities for studying neuronal properties and development1,2. Of the ...

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 Rat Brain Aggregate Cultures for Neuron and Glia Development Studies

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

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

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

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

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 ...

Intranuclear Microinjection of DNA into Dissociated Adult Mammalian Neurons

Primary neuronal cell cultures are valuable tools to study protein function since they represent a more biologically relevant system compared to immortalized cell lines. However, the post-mitotic nature of primary neurons prevents effective heterologous protein expression using common procedures such as electroporation or chemically-mediated transfection. Thus, other techniques must be employed in order to effectively express proteins in these non-dividing cells.
In this article, we describe the steps required to perform intranuclear injections of cDNA constructs into dissociated adult sympathetic neurons. This technique, which has been applied to different types of neurons, can successfully induce heterologous protein expression. The equipment essential for the microinjection procedure includes an inverted microscope to visualize cells, a glass injection pipet filled with cDNA solution that is connected to a N2(g) pressure delivery system, and a micromanipulator. The micromanipulator coordinates the injection motion of microinjection pipet with a brief pulse of pressurized N2 to eject cDNA solution from the pipet tip. 
This technique does not have the toxicity associated with many other transfection methods and enables multiple DNA constructs to be expressed at a consistent ratio. The low number of injected cells makes the microinjection procedure well suited for single cell studies such as electrophysiological recordings and optical imaging, but may not be ideal for biochemical assays that require a larger number of cells and higher transfection efficiencies. Although intranuclear microinjections require an investment of equipment and time, the ability to achieve high levels of heterologous protein expression in a physiologically relevant environment makes this technique a very useful tool to investigate protein function.

Direct intranuclear injection of cDNA is an effective transfection technique for post-mitotic cells. This method provides high levels of heterologous protein expression from single or multiple cDNA constructs and enables protein function to be studied in a physiologically relevant environment with a variety of single cell assays.