Preparações antes do dia da cultura: <ul><li> Preparar a solução de dissecação estéril (DS). </li><li> Prepare NBM/B27 (Mediom Neurobasal com suplementos B27). </li><li> Autoclave DDH 2 O e esterilizar coversilps vidro, se necessário. </li><li> Brasão pratos de cultura de tecidos ou lamínulas de vidro com poli-D-lisina. </li></ul> Poli-D-lisina Revestimento: Prepare um dia antes de cultivo em condições estéreis. <ul><li> Alíquota de descongelar PDL 10X e colocar no gelo. </li><li> Adicionar 9 ml de água ultra-filtrado estéril para 1 ml de PDL e misture bem (1X). </li><li> Revestimento com superfícies 1X PDL durante a noite a temperatura ambiente da seguinte forma: <ul><li> lamínulas de vidro (Bellco Cat Glass, Inc. # 1943-00012.): 75 mL / lamela OR </li><li> 24 placa de cultura bem: 300 l / poço OR </li><li> 35 milímetros placa de cultura: 1ml/dish </li></ul></li><li> Enxágüe com água estéril 5X (use pipetas Pasteur estéril para aspirar líquido). </li><li> Remover a água até a superfície está completamente seca. </li></ul> Procedimento de cultivo (feito em capela de fluxo laminar em condições estéreis) <ol><li> Cortar bloco de agar e cola-lo para o bloco de apoio do Vibraslicer micrótomo (Campden Instruments Ltd.), utilizando cola super. </li><li> Ao usar fase embrionária tardia (E17-18) fetos de rato, barragem eutanásia, retirada do útero e livre fetos individuais de saco embrionário. Fetos colocar em placa de Petri estéril e continuar como descrito abaixo. </li><li> Decapitar feto de rato ou cachorro (siga as orientações aprovadas pelo seu Animal Care Institucional e Comitê de Uso). </li><li> Retire a pele e crânio e do cérebro lugar em papel de filtro Whatman disco numa placa de Petri 60 milímetros cheio de DS frio. </li><li> Cortar o cerebelo usando uma lâmina estéril. </li><li> Pick up cérebro usando uma espátula e drenar o excesso de fluido em papel de filtro, em seguida, transferir cérebro para Vibraslicer bloco de apoio e cole no lugar do cérebro (lado caudal acima e parte ventral de frente para agar). </li><li> Encher a câmara com o DS frio. Seletor de velocidade para 8-9. </li><li> Corte 200-400 seções M, a partir do bulbo olfativo. Uma vez que a lâmina da Vibraslicer entra no córtex, começar a cortar 600μm cortes coronais. Um cérebro E17-18 do mouse normalmente produz 3-4 fatias utilizável, enquanto que um cérebro de camundongo P0 rendimentos 4-5 fatias utilizável. </li><li> Transferência de fatias do cérebro a 35 milímetros prato petri rotulados DS 2 com fim de reverter un-conectado 10 ml de vidro pipeta Pasteur. </li><li> Dissecar córtex e remover meninges utilizando agulhas de vidro retirado tubos capilares. </li><li> Cortar cascas cortical em pequenos pedaços, 0,5-1 mm de comprimento. </li><li> Filtro ES através de filtro de 0,2 mm acoplada a uma seringa cc 5 em uma placa de Petri 35 mm. </li><li> Transferir os pedaços de tecido cortical para placa de Petri contendo ES filtrada. </li><li> Incubar a 37 ° C por 30 min. </li></ol> Nesse meio tempo: <ol><li> Limpar Vibraslicer capô, e ferramentas de dissecação. </li><li> Adicionar 5 ml de NBM/B27 quente em uma placa de Petri 60 mm. </li></ol> Depois de 30 min. de incubação: <ol><li> Transferência de tecido para 10 ml DS. Vamos se contentar com 1 min. </li><li> Transferência de tecido para redemoinhos primeiro tubo Hi e deixar assentar por 2-3 min. </li><li> Transferência para Hi segundo. Repita como indicado acima. </li><li> Transferência para Li em primeiro lugar. Repita como indicado acima. </li><li> Li transferência para segundo. Repita como indicado acima. </li><li> Li transferência para terceiros. Repita como indicado acima. </li><li> Transferência de tecido para o prato com 60 milímetros de mídia. </li></ol> Sob o microscópio de dissecção: <ol><li> Restos de tecido limpo e triturar cada peça suavemente passa através de uma pipeta de vidro puxado (use a diminuir tamanhos de furo) para soltar o tecido. </li><li> Transferência de suspensão de células a 15 ml tubo cônico contendo o resto da NBM + B27 (Adicione um total de 13 ml para uma placa de 24 poços ou 11 ml por 5 - 35. Pratos milímetros Misture suavemente e espere grandes pedaços de tecido para resolver). </li><li> Suspensão de transferência de células (0,5 ml / poço de uma placa de 24 poços ou 2 ml / 35 placa de cultura mm). </li><li> Ao usar lamínulas de vidro, adicionar 80 mL de suspensão celular por lamínula e permitir uma hr em cultura de tecidos incubadora para as células a aderirem antes de inundar a câmara com mais NBM/B27 mídia. </li><li> Manter culturas a 37 ° C e 5% CO 2. </li></ol> Dia Seguinte 24 placa assim: Alimente-se bem com 0,5 de células não-neuronais ml condicionado NBM/B27 médio (cNBM/B27; Protocolo para a preparação de cNBM/B27 médio aparece abaixo) Pratos 35mm: Substitua 0,5 ml com cNBM/B27 médio. Manter a cultura através da substituição de 0,5 ml de meio com cNBM/B27 novo a cada 2-3 dias. Embora os meios de cultura não promove a proliferação de células glia, as culturas podem ser tratados com 5m FDU (5 Fluoro-2&#39;-deoxiuridina, Sigma F0503) no dia 05/03 na cultura para reduzir ainda mais o número de células gliais, se necessário. SET<html> -SE para dissecção e Cultura Esterilizar instrumentos de dissecação em EtOH 70% ou autoclave eles: <ul><li> Tesoura (empresas de médio e pequeno) Espátulas (empresas de médio e pequeno) </li><li> Pinças lâmina de barbear (empresas de médio e pequeno) </li><li> Lâmina para vibraslicer banho de tampão </li><li> Cunha metálica para suporte de lâmina para o cérebro </li></ul> Outros materiais: <ul><li> Placas de Petri (60 mm) </li><li> Placas de Petri (35 mm) </li><li> Papel-filtro </li><li> Pipeta de vidro de 10 ml </li><li> Pipetas estéreis descartáveis, 5,10 e 25 ml </li><li> Seringa filtro, 0,2 m </li><li> Seringa de 5 cc </li><li> Tubos de centrífuga, 15 ml </li><li> Estéril Pasteur de vidro pipetas </li><li> PDL revestido placas de cultura ou de lamínulas de vidro </li></ul> Rótulo seis tubos de centrífuga de 15 ml e siga preparação: Tubo # 1: solução enzimática: Adicionar 50 U de papaína (Worthington LS 03126) a 5 DS ml contendo: <ul><li> 100μl de L-cisteína (0,8 mg) </li><li> 7 mL NaOH 0,1 N </li><li> 50 APV mL (5mm) </li></ul> Deixar solução em temperatura ambiente para limpar. Note-se que solução enzimática aparecerá &quot;nublado&quot; em primeiro lugar e precisa clara antes de usar. Tubo # 2 e # 3: Hi inibidor da enzima: 3 ml + 300 ml DS BSA / Ti + 30 mL APV (5mm). Misture com cuidado para evitar bolhas, em seguida, dividir em duas alíquotas de 1,5 ml. Tubo # 4 - # 6: inibidor da enzima Low: 8 ml DS + 80 mL BSA / Ti + 80 mL APV (5mm). Misture com cuidado para evitar bolhas, divida em três alíquotas de 2,6 ml. Colocar os tubos numerados # 2 a # 6 em gelo. Deixe tubo # 1 (solução enzimática) em temperatura ambiente. SOLUÇÕES e alíquotas <ul><li> Uma solução (Buffered Saline) Sigma Formula wt 500 ml [conc.] </li><li> Cloreto de Sódio NaCl S-9625 58,45 80.0g 137mm </li><li> Cloreto de potássio KCl P-4504 74,56 4,0 G 5.4mm </li><li> Fosfato de sódio dibásico anidro Na 2 HPO 4 S-0876 142,0 0,24 g 0.17mm </li><li> Potássio fosfato monobásico anidro KH 2 PO 4 P-5379 136,09 0,3 g 0,22 mm </li></ul> Pesar todos os ingredientes e misture até dissolver com 400 ml de água ultra-filtrada. Trazer um volume final de 500 ml. Coloque em uma garrafa limpa e autoclave. Armazenar a 4 ° C e etiqueta &quot;Solução DS A&quot;. <ul><li> Solução B (Hepes) Sigma Formula wt 250 ml [conc.] </li><li> Hepes Hepes H-3375 283,3 20.97g 9,9 mm </li></ul> Adicionar ultra-água filtrada até 200 ml. Misture até dissolver e trazer um volume final de 250 ml. Coloque em uma garrafa limpa e autoclave. Armazenar a 4 ° C e etiqueta &quot;Solução DS B&quot;. <ul><li> Solução de trabalho 500 ml [conc.] </li><li> Ultra água filtrada 400 ml </li><li> A solução estoque de 25 ml </li><li> Solução estoque B 14 ml </li><li> D (+) glicose-Sigma G-8270 3,0 g 33.3mM </li><li> Sacarose Sigma S-0389 7,5 g 43,8 mM </li></ul> Ajustar o pH para 7,4 com NaOH 1N. Trazer um volume final de 500 ml com água filtrada ultra. Decantar dentro de uma garrafa de vidro limpa e autoclave. Armazenar a 4 ° C. &quot;Solução Dissecando&quot; rótulo. Poli-D-lisina de preparação: <ol><li> Preparar a solução estoque 10x de poli-D-lisina (PDL; Sigma P-7280), em H 2 O estéril em 1mg/ml. </li><li> Faça alíquotas 1,0 ml. Esta solução pode ser armazenada a -20 ° C por até 3 meses. </li></ol> MÍDIA <ol><li> Adicionar 10ml de B-27 Suplemento (Gibco 17504-044) para frasco de 500 ml NBM (Medium Neurobasal, Gibco 21103-049). </li><li> Fazer 40 ml alíquotas e armazenar a 4 ° C. </li></ol> APV <ol><li> Adicionar 10 ml H 2 O estéril para o frasco de 10 mg APV (2-Amino-5-phosphonopentanoic ácido, Sigma A-5282) e misture bem. </li><li> Prepare 180 mL alíquotas e armazenar a -20 ° C. </li></ol> BSA / Ti <ol><li> Dissolver 1 g BSA (albumina bovina. Sigma A-7030) e 1 g inibidor de tripsina (Sigma T-9253) em 10 ml DS. </li><li> Ajustar o pH para 7,4 com NaOH 1N. </li><li> Esterilizar por filtração através de filtro de seringa de 0,2 mM. </li><li> Dividem-se em 400 mL alíquotas e armazenar a -20 ° C. </li></ol> L-cisteína Em um tubo Eppendorf dissolver 0,6 mg L-Cisteína (Sigma C-7755) em 200 mL DS. AGAR (4%) <ol><li> Dissolver 4 g de Agar Bacto (Difco 0140-01) em 100 ml de água estéril. Manter a 4 ° C até ser necessário para a cultura. </li><li> Agar microondas para derreter e encher um 35 milímetros placa de Petri. Vamos sentar para esfriar e polimerizar. </li></ol> Papaína (Worthington LS 03126) e_title &quot;&gt; não neuronais CULTURAS EM GARRAFAS CULTURA NUNC GARRAFA DE REVESTIMENTO DE CULTURA (4 garrafas Nunc) <ol><li> Adicionar 9 ml de água estéril para cada um dos três tubos de PDL 10X fazer PDL 1X. </li><li> Transferência de 30 ml 1X PDL na primeira garrafa. </li><li> Mover-se ligeiramente até que todas as superfícies de crescimento são cobertos. Deixe descansar por 1 min. </li><li> Transferência 1x PDL a segunda garrafa. Deixe descansar por 1 min. </li><li> Repita com as garrafas de terceira e quarta. </li><li> Lave bem todas as quatro garrafas 2X com 150 ml estéril H 2 O. </li></ol> PREPARE solução enzimática (ES): <ol><li> Em um tubo de 0,6 ml de peso centrífuga para fora 0,8 mg L-Cisteína (Sigma C7755), adicione 150ml DS e vortex até que os cristais são dissolvidos. </li><li> Transferir 5 ml DS a um tubo cônico de 15 ml, em seguida, adicione 150ml L-Cisteína. </li><li> Adicionar 50 unidades de papaína. (Worthington LS 03126) </li><li> Adicionar 7ml 0.1N NaOH. </li></ol> PREPARE MEM (GIBCO # 11090-081) <ol><li> Retire 65 ml da garrafa MEM 500 ml. Economize 10 ml para preparar glicose. </li><li> Adicionar 5 ml Pen / Strep (Gibco # 15070-063). </li><li> Adicionar 10 ml de glicose 1M (Sigma G-8270) (1,8 ml g / 10 MEM) </li><li> Adicionar 50 ml de soro fetal bovino (Gibco # 16140-071) ou bovina Calf Serum (Omega BC-04). </li><li> Misture bem alíquota, e armazenar a 4 ° C. </li></ol> SUPLEMENTO MEDIUM/B-27 NEUROBASAL (NBM/B27) NBM, 500 ml (Gibco # 21103-049); B-27 (50X), 10 ml (Gibco # 17504-044) <ol><li> Adicionar todo o conteúdo do frasco B-27 para a garrafa NBM e misturar. </li><li> Alíquotas e armazenar a 4 ° C. </li></ol> DISSECTION do tecido cerebral <ol><li> Seca dissecar ferramentas de EtOH 70% antes da dissecção. </li><li> Transfira 10 ml DS em cada um tubo de 15/03 ml. </li><li> Selecione filhotes mouse (P0 - P3), você precisará de três cérebros para 4 garrafas. </li><li> Adicionar DS fria a 35 milímetros prato de petri. </li><li> Decapitar rato filhote e dissecar o cérebro. </li><li> Cérebros colocar na placa de petri contendo DS frio. </li><li> Pique em pedaços de tecido, pequeno o suficiente para passar através de uma pipeta de vidro. </li></ol> ENZIMA DISSOCIAÇÃO <ol><li> Remover o DS tanto quanto possível, a partir do prato com uma pipeta Pasteur. </li><li> Filtro ES através de uma seringa 0,2 m de filtro e 5cc. </li><li> Lugar ES no prato com o tecido. </li><li> Incubar tecido a 37 ° C incubadora (CO 2 livre) por 30 min. </li></ol> LAVAGEM DE TECIDOS <ol><li> Tecidos de transferência com pipeta de vidro para o tubo DS primeiro, certificando-se de obter o ES mínimo possível. </li><li> Centrifugar por 30 segundos a 1500 rpm. </li><li> Transferência de tecidos e repetir o procedimento mais duas vezes. </li></ol> TRITURAÇÃO <ol><li> Coloque 38 ml de MEM / FBS em um tubo de centrífuga de 50ml </li><li> Adicionar 3 ml de MEM / FBS a um prato de 35 mm e tecido transferir para esta placa de Petri. </li><li> Dissociar o tecido suavemente triturando-lo através de uma ponta de pipeta 1000μl eppendorf. </li><li> Transferência de suspensão de células para o tubo contendo 38ml de MEM / FBS e misture bem. </li></ol> PLATING <ol><li> Suportar a cultura garrafas em posição vertical para obter a mesma quantidade de suspensão de células e meios de comunicação em cada garrafa. </li><li> Coloque 90 ml de MEM / FBS em cada frasco de cultura. </li><li> Adicionar 10 ml de suspensão de células em cada garrafa. </li><li> Label: Non-Neuronal, iniciais e data. Incubar a 37 ° C e 5% CO 2. </li></ol> ALIMENTAÇÃO <ol><li> No dia 3 ou 4, as células de alimentação com MEM quente / FBS (decantar todos os meios de comunicação e substituí-la por 100 ml de MEM / FBS). Culturas pode levar 7-10 dias para se tornar confluentes. </li><li> No dia 10/07 alterar a mídia para NBM/B27. </li><li> No dia seguinte, recolher os meios de comunicação. Filtro com 0.22μm </li><li> Faça alíquotas de 40 ml e alimentar a cultura com MEM / FBS. </li><li> Próximos 2 ou 3 dias, a mudança de mídia para NBM/B27 </li><li> No dia seguinte, coletar e repita o procedimento. </li><li> Mantenha coleta de mídia para aprox. Duas semanas. </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

Este vídeo irá guiá-lo através do processo de geração de culturas cortical neuronal de embriões tarde e cérebro de camundongos pós-natal precoce. Essas culturas podem ser usados ​​para uma variedade de aplicações, incluindo imunocitoquímica, bioquímica, eletrofisiologia, imageamento de cálcio e sódio, proteína e / ou RNA isolamento. Estas culturas também fornecer uma plataforma para estudar o desenvolvimento neuronal de animais transgênicos que carregam uma mutação genética tarde embrionárias ou pós-natal letal. O procedimento é relativamente simples, requer alguma experiência na técnica de cultura de tecidos e não deve demorar mais do que 2-3 horas, se você estiver devidamente preparado. Uma separação cuidadosa da casca cortical do trato fibras tálamo-cortical vai reduzir o número de gravidezes indesejadas células não-neuronais. Para aumentar a produção de células neuronais triturar os pedaços do tecido cortical delicadamente após a etapa de incubação enzimática. Isto é imperativo, uma vez que evita lesões desnecessárias para as células e prematura morte celular neuronal. Uma vez que estas culturas são mantidos na ausência de células alimentadoras glia, eles também oferecem uma vantagem adicional de culturas em crescimento enriquecido em neurônios.

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

Preparation of Dissociated Mouse Cortical Neuron Cultures

Este vídeo mostra um procedimento para a geração de culturas de neurônios a partir de embrião tarde e córtex do rato pós-natal precoce. Essas culturas podem ser usados ​​para imunocitoquímica, bioquímica, eletrofisiologia, cálcio e sódio de imagem e fornecer uma plataforma para estudar o desenvolvimento neuronal de animais transgênicos que carregam uma mutação genética pós-natal letal.

Preparação de Dissociated rato Culturas Neuron Cortical

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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JoVE Journal

Biology

43.2K visualizações.  University of California, Irvine (UCI). Este vídeo mostra um procedimento para a geração de culturas de neurônios a partir de embrião tarde e córtex do rato pós-natal precoce. Essas culturas podem ser usados ​​para imunocitoquímica, bioquímica, eletrofisiologia, cálcio e sódio de imagem e fornecer uma plataforma para estudar o desenvolvimento neuronal de animais transgênicos que carregam uma mutação genética pós-natal letal.

Vídeo: 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.

Preparação de culturas de neurônios a partir de embriões Midgastrula Stage Drosophila

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

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.

Rato Epidérmico Neural Stem Cell Crest (EPI-NCSC) Culturas

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.

Principal Dissociated Midbrain dopamina culturas de células de recém-nascidos de roedores

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.

Preparação de 2-dGuo-Tratada Culturas Organ Thymus

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.

Preparação e manutenção dos neurônios da raiz dorsal Gânglios em culturas 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 Cultura de Pós-Natal do rato Cerebelar Granule Células Progenitoras Neuron e Neurônios

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.

Elaboração de culturas de células primárias hematopoéticas de medula óssea de camundongos para Eletroporação

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.

Preparação das Culturas cérebro de um rato Aggregate para Neuron Glia e Estudos de Desenvolvimento

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.

Preparação de Aplysia Sensório-motor Neuronal culturas de células

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.