Este estudo foi apoiado pela Fundação Nacional de Ciência Natural da China (Grant nº 81673676) e pelo Dongguan Science and Technology Bureau (Grant nº 2019622101002). Os autores agradecem à Dra.

Todos os protocolos experimentais e procedimentos animais cumpriram as diretrizes do princípio ético do Conselho Nacional de Pesquisa.
1. Preparação de materiais
<ol><li>Esterilize todos os instrumentos e ddH2O usando uma autoclave antes de realizar o experimento. Os instrumentos incluem, mas não se limitam a tesouras cirúrgicas, tesoura oftálmica, fórceps, divisor de núcleos de colheres, pratos de vidro (60-100 mm de diâmetro) e quebra-vidros.</li>
	<li>Prepare a solução Krebs da seguinte forma(Tabela 1): Dissolva todos os produtos químicos juntamente com ddH2O antes de usar. Dissolva o CaCI2 separadamente e adicione lentamente na solução mista enquanto mexe para evitar sedimentos.</li>
	<li>Prepare a mídia Rinse, que consiste em mídia F12 com 10% de soro bovino fetal (FBS) e 1% antibiótico/antimíctico (100x). Adicione 5 mL de FBS e 0,5 mL de antibiótico/antimíctico a 44,5 mL de mídia F12.</li>
	<li>Prepare a mídia de neurônioS A, que compreende a mídia neurobásica A com 2% B-27, 1% FBS, 1% L-glutamina, 1% antibiótico/antimíctico (100x) e fator neurotrófico derivado de glia de 0,1% (GDNF). Adicione 200 μL de B-27, 100 μL de FBS, 100 μL de L-glutamina, 100 μL de antibiótico/antimíctico (100x) e 10 μL de GDNF (10 μg/mL) a 9,5 mL de mídia neurobásal A. NOTA: Prepare a mídia de neurônios A dentro de uma semana de uso para garantir o frescor de B-27, L-glutamina e GDNF.</li>
	<li>Prepare a mídia de neurônio B usando o mesmo procedimento que a preparação da mídia de neurônios A, mas sem adicionar FBS.</li>
	<li>Prepare a solução de digestão 1 dissolvendo 20 mg de colagenase tipo II, 6 mg de albumina de soro bovino e 200 μL de antibiótico/antimíctico (100x) em 10 mL de solução krebs estável em oxigênio.</li>
	<li>Prepare a solução de digestão 2 diluindo 1 mL de 0,25% de trippsina com 4 mL da solução de sal balanceada da Hank.</li>
	<li>Prepare um prato com tampas revestidas.<ol>
<li>Use fórceps em um banco de fluxo laminar ao colocar tampas de vidro em uma placa de cultura de 48 poços.</li>
		<li>Diluir a poli-D-lysina com ddH2O a uma concentração de 0,1 mg/mL como estoque de poli-D-lysina. Armazene o estoque a -20 °C e descongele antes de usar.</li>
		<li>Adicione 40 μL de material de poli-D-lisina em cima de cada deslizamento de cobertura, e incubar a solução em temperatura ambiente por 10 minutos. NOTA: Para diferentes placas com diferentes áreas basais, ajuste a concentração para 4 μg/cm2. Por exemplo, se a área basal de um poço de uma placa de 24 poços é de 2 cm2,então devemos transferir 80 μL de estoque de poli-D-lysina em um poço. Cada cm2 conteria 4 μg de poli-D-lysine.</li>
		<li>Remova a poli-d-lisina na placa de 48 poços e enxágue os deslizamentos com ddH2O três vezes.</li>
		<li>Seque a placa em um capô de fluxo laminar por pelo menos 30 minutos para garantir que a placa esteja anidro.</li>
		<li>Armazene a placa a 4 °C antes do revestimento de laminina durante no máximo 1 dia. Armazenar a placa a −20 °C é preferível para armazenamento a longo prazo.</li>
		<li>Descongele laminina a 4 °C. Diluir laminina com ddH2O a uma concentração de 50 μg/mL como estoque de laminina. Armazene o estoque a -20 °C e descongele a 4 °C antes de usar.</li>
		<li>Use uma pipeta para transferir 100 μL de laminina diluída para o topo de cada deslizamento de cobertura e incubar laminina a 4 °C por 1h. NOTA: Para diferentes placas com diferentes áreas basais, ajuste a concentração para 5 μg/cm2. Por exemplo, se a área basal de um poço de uma placa de 24 poços for de 2 cm2, então transfira 200 μL de laminina diluída em um poço. Cada cm2 conteria 5 μg de laminina.</li>
		<li>Remova a solução de laminina e enxágue as tampas com ddH2O uma vez. Realize esta operação na borda do deslizamento para evitar raspagem. NOTA: As tampas revestidas podem ser armazenadas a 4 °C durante 2 semanas no máximo.</li>
	</ol>
</li>
</ol>2. Colheita da bexiga
<ol><li>Obter ratos Sprague-Dawley de cinco semanas de idade.</li>
	<li>Infundir carbogen (95% oxigênio, 5% CO2) na solução Krebs por pelo menos 30 minutos em um banho de gelo para atingir um estado estável de oxigênio e nível de pH.</li>
	<li>Após a eutanásia por deslocamento cervical, mergulhe os ratos em 75% de etanol para 30 s para esterilização.</li>
	<li>Coloque ratos em uma toalha cirúrgica esterilizada e exponha seu abdômen. Abra a cavidade abdominal e revele a bexiga com um conjunto de tesouras e fórceps.</li>
	<li>Levante a bexiga suavemente e corte a bexiga do pescoço da bexiga com outro conjunto de tesouras e fórceps para evitar contaminação cruzada. Coloque a bexiga rapidamente em solução krebs estável em oxigênio frio para melhorar a sobrevivência celular. NOTA: Uma vez que a bexiga é removida, realize as seguintes operações rapidamente para melhorar a perspectiva de sobrevivência dos neurônios.</li>
	<li>Adicione a solução Krebs em três pratos de vidro e quebra-vidros.</li>
	<li>Prepare pratos de vidro e quebra-vidros com solução Krebs em um banho de gelo para pré-fabricação.</li>
	<li>Marque esses recipientes com números 1-3 correspondentemente para evitar confusão.</li>
	<li>Emparelhe cada prato de vidro com fórceps e um divisor de núcleos colheres.</li>
	<li>No prato de vidro 1, abra a bexiga com uma tesoura oftálmica, e desdobre-a com fórceps e um divisor de núcleos de colheres.</li>
	<li>Enxágüe a bexiga no quebra-vidro 1 e coloque-a no prato de vidro 2.</li>
	<li>Elimine a gordura aderente na superfície do tecido usando as fórceps e a tesoura oftálmica no prato de vidro 2.</li>
	<li>Enxágüe a bexiga no quebra-vidro 2 e coloque-a no prato de vidro 3.</li>
	<li>Raspe suavemente a bexiga usando as fórceps e o divisor do núcleo das colheres no prato de vidro 3 para remover os acessórios exógenos.</li>
	<li>Enxágüe a bexiga no disjuntor de vidro 3, transfira a bexiga para um tubo de centrífugas de 15 mL com 14 mL de solução krebs fria, e gire a amostra por 1 min a 356 x g e 4 °C.</li>
	<li>Repita a etapa anterior duas vezes em dois outros tubos com solução Krebs para reduzir a contaminação.</li>
</ol>3. Digestão da bexiga de dois passos
<ol><li>Transfira a bexiga do tubo centrífuga para um frasco de 2 mL contendo 1 mL de solução de digestão 1. Use uma tesoura oftálmica para cortar a bexiga em pequenos pedaços (menores que 1 mm) na solução.</li>
	<li>Misture a solução da bexiga com 9 mL de solução de digestão 1 em um prato de cultura celular estéril (100 mm de diâmetro). Realize a digestão passo 1 em uma incubadora de agitação por 1h abaixo de 5% de CO2, 37 °C e 200 rpm.</li>
	<li>Após a digestão do passo 1, centrifugar a solução a 356 x g a 4 °C por 8 min.</li>
	<li>Coloque a solução de digestão 2 em um banho de água de 37 °C para pré-aquecimento.</li>
	<li>Após a centrifugação, remova o sobrenante que contém a solução de digestão 1, e colmeia o sedimento celular. Algum líquido restante é permitido. A remoção completa da solução pode promover a perda celular.</li>
	<li>Misture sedimentos celulares com solução de digestão quente 2 em um tubo de centrífugas de 15 mL e agite a mistura enquanto digere em um banho de água de 37 °C por 5 min. Não exceda 7 minutos de digestão do passo 2 ou os neurônios perecerão.</li>
	<li>Após a digestão, desative imediatamente a trippsina na mistura com 10 mL de mídia de lavagem a frio. NOTA: Realize as seguintes etapas em 0 °C–4 °C. Um banho de gelo poderia fornecer tal condição.</li>
	<li>Colher o sedimento celular após centrifugação a 356 x g a 4 °C por 8 min. Remova o máximo de mídia possível porque a trippsina restante é prejudicial ao crescimento celular.</li>
	<li>Sedimento resuspend com 3 mL de mídia de neurônios suavemente. Certifique-se de que as bolhas de ar não sejam geradas na solução que contém células para uma alta taxa de sobrevivência.</li>
	<li>Filtre a mídia da mistura através de um coador de células de 70 μm em um tubo centrífuga de 50 mL.</li>
	<li>Mantenha o filtrado em um shaker a 30 rpm em um banho de gelo por 30 minutos. Esta etapa não é necessária, mas recomendada.</li>
	<li>Coletar células por centrifugação a 356 x g a 4 °C por 8 min, e resuspensar suavemente as pelotas celulares em 1 mL de mídia neurônio A.</li>
	<li>Adicione 500 μL de mistura celular em cada poço da placa de 48 poços preparada.</li>
	<li>Células de cultura em uma incubadora a 37 °C e 5% DE CO2.</li>
	<li>Substitua toda a mídia por mídia de neurônios B em 1h para fornecer uma cultura livre de soro.</li>
	<li>Troque metade da mídia de neurônios B a cada 3 dias. NOTA: Os neurônios estão prontos para experimentos imunocitômicos após 5-7 dias de cultura.</li>
</ol>

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Os autores não declaram nenhum grande conflito de interesses.

Preparação da placa O uso de tampas de vidro em placas de cultura de 6, 12 ou 48 poços para experimentos de imagens imunofluorescentes ou de cálcio é uma operação econômica e poupadora de amostras. As células crescem bem em placas sem tampas durante a preparação das culturas celulares primárias. Portanto, as tampas são dispensáveis em experimentos, como a mancha ocidental ou a reação em cadeia de polimerase. Além disso, o revestimento é um passo necessário antes de emplacamento de c...

O trato urinário inferior tem duas funções principais: armazenamento periódico de urina e micturição1. O sistema nervoso do trato urinário inferior (LUTNS) controla essas funções e é delicado e suscetível a muitas neuropatias, que podem ser inatas (porfiria), adquiridas (doença de Lyme), secundárias aos estados da doença (cistopatia diabética), induzida por drogas (cistite hemorrágica), cirurgia causada (ressecção abdominoperineal) ou lesão causada (lesão medular traumática)2,3,4,5,6,7. Em estudos fisiológicos/patológicos, experimentos in vivo e in vitro são igualmente importantes. Enquanto a pesquisa in vivo em LUTNS tem sido conduzida em níveis de órgãos, celulares e moleculares há algum tempo, a pesquisa in vitro sobre neurônios primários da bexiga urinária é quase inexistente8,9. Embora o presente estudo seja limitado, esperamos ser pioneiros em pesquisas nessa área para que outros pesquisadores possam melhorá-la. Dessa forma, essa cocultura pode levar a uma compreensão celular da disfunção fisiológica em fenótipos, como a disfunção do neurônio da bexiga.
Em contraste com os músculos entéricos com uma direcionalidade clara das células musculares em camadas discretas, os músculos da bexiga são desorganizados10. Portanto, em vez de descascar a camada externa da bexiga, este método propõe digerir toda a bexiga para reduzir a dificuldade de operação e encurtar o tempo de pré-processamento para uma alta taxa de sobrevivência celular.
Seguindo este método, podemos obter uma cultura mista de neurônios e outras células. As outras células são indispensáveis porque sua presença imita um ambiente in vivo11. Além disso, essas células fornecem as substâncias que não estão disponíveis no meio.
Este método envolve dois passos para a digestão. Primeiro, a colagem tipo II é usada para hidrolisar colágeno, seguido de trippsina, para dissociar o tecido nas células10. Dessa forma, os tecidos da bexiga são dispersos em células únicas e, em seguida, crescem relativamente independentes. Quando a cultura dos neurônios amadurece, os neurônios podem ser usados para imagens ou ensaios funcionais.

<table border="0" cellpadding="0" cellspacing="0" style="width:1061px;" width="1061">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:368px;">0.25% trypsin</td>
			<td style="width:279px;">Gibico</td>
			<td style="width:119px;">15050065</td>
			<td style="width:296px;">Enzyme digestion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">48-well culture plate</td>
			<td>Corning</td>
			<td style="width:119px;">3548</td>
			<td>Coating dish</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">antibiotic/antimycotic</td>
			<td>Gibico</td>
			<td>15240062</td>
			<td>Culture media/Rinse media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Glial Fibrillary Acidic Protein Antibody</td>
			<td>Santa Cruz</td>
			<td>sc-33673</td>
			<td>ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">B-27</td>
			<td>Gibico</td>
			<td>17504044</td>
			<td>Culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BSA Fraction V</td>
			<td>Gibico</td>
			<td>332</td>
			<td>Enzyme digestion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Choline Acetyltransferase Antibody</td>
			<td>Abcam</td>
			<td>ab18736</td>
			<td>ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CO2 Incubator</td>
			<td>Heraeus</td>
			<td>B16UU</td>
			<td>Cells culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase type II</td>
			<td>Sigma</td>
			<td>2593923</td>
			<td>Enzyme digestion</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;">DMEM/F-12</td>
			<td>Gibico</td>
			<td>11330032</td>
			<td>Rinse media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DYNLL2 Antibody</td>
			<td>Santa Cruz</td>
			<td>sc-13969</td>
			<td>ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum</td>
			<td>Gibico</td>
			<td>10100147</td>
			<td>Culture media/Rinse media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forceps</td>
			<td>Shanghai Jin Zhong Medical Devices</td>
			<td>1383</td>
			<td>10 cm; Sterile operation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass breakers</td>
			<td>Huan Qiu Medical Devices</td>
			<td>1101</td>
			<td>50 ml; Sterile operation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass coverslips</td>
			<td>WHB Scientific</td>
			<td>WHB-48-CS</td>
			<td>Coating dish</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass dishes</td>
			<td>Huan Qiu Medical Devices</td>
			<td>1177</td>
			<td>100 mm; Sterile operation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat Anti-Rat IgG(H+L), Mouse ads-Alexa Fluor 488</td>
			<td>Southernbiotech</td>
			<td>3050-30</td>
			<td>ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat Anti-Rat IgG(H+L), Mouse ads-Alexa Fluor 555</td>
			<td>Southernbiotech</td>
			<td>3050-30</td>
			<td>ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hoechst 33342</td>
			<td>BD</td>
			<td>561908</td>
			<td>ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Laminar flow bench</td>
			<td>Su Jie Medical Devices</td>
			<td>CB 1400V</td>
			<td>Sterile operation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Laminin</td>
			<td>Sigma</td>
			<td>L2020</td>
			<td>Coating dish</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-glutamine</td>
			<td>Gibico</td>
			<td>25030081</td>
			<td>Culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MAP-2 Antibody</td>
			<td>Affinity</td>
			<td>AF5156</td>
			<td>ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Murine GDNF</td>
			<td>Peprotech</td>
			<td>AF45044</td>
			<td>Culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Neurobasal-A Medium</td>
			<td>Gibico</td>
			<td>10888022</td>
			<td>Culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ophthalmic scissors</td>
			<td>Shanghai Jin Zhong Medical Devices</td>
			<td>J21010</td>
			<td>12.5 cm; Sterile operation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pipettes</td>
			<td>Eppendorf</td>
			<td style="width:119px;">3120000240</td>
			<td>100-1000 ul; Reagent and sample pipetting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pipettes</td>
			<td>Eppendorf</td>
			<td style="width:119px;">3120000267</td>
			<td>10-100 ul; Reagent and sample pipetting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly-D-lysine</td>
			<td>Sigma</td>
			<td>P7280</td>
			<td>Coating dish</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Refrigerated centrifuge</td>
			<td>Ping Fan Instrument</td>
			<td>TGL-16A</td>
			<td>Enzyme digestion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Shaking incubator</td>
			<td>Haimen Kylin-Bell Lab Instruments</td>
			<td style="width:119px;">T8-1</td>
			<td>Enzyme digestion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SLC17A9 Antibody</td>
			<td>MBL International</td>
			<td>BMP079</td>
			<td>ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spoons nucleus divider</td>
			<td>Shanghai Jin Zhong Medical Devices</td>
			<td>YZR030</td>
			<td>12 cm; Sterile operation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Substance P Antibody</td>
			<td>Santa Cruz</td>
			<td>sc-58591</td>
			<td>ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Surgical scissors</td>
			<td>Shanghai Jin Zhong Medical Devices</td>
			<td>J21130</td>
			<td>16 cm; Sterile operation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Surgical towel</td>
			<td>Fu Kang Medical Devices</td>
			<td>5002</td>
			<td>40 x 50 cm; Sterile operation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Synapsin-1 Antibody</td>
			<td>CST</td>
			<td>5297T</td>
			<td>ICC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tubulin beta Antibody(&#946;-III-tubulin)</td>
			<td>Affinity</td>
			<td>AF7011</td>
			<td>ICC</td>
		</tr>
	</tbody>
</table>

isolation and culture of primary neurons and glia from adult rat urinary bladder

O trato urinário inferior tem duas funções principais: armazenamento periódico de urina e micturição; essas funções são mediadas através da neuroregulação central e periférica. Embora tenham sido realizadas extensas pesquisas sobre o sistema nervoso do trato urinário inferior, a maioria dos estudos tem se concentrado na cultura primária. Este protocolo introduz um método para o isolamento e cultura dos neurônios da bexiga e da glia dos ratos de Sprague-Dawley. Neste método, os neurônios e a glia foram incubados em uma incubadora de 37 °C, 5% de CO2 por 5-7 dias. Como resultado, eles cresceram em formas maduras adequadas para experimentos de imunofluorescência subsequentes relacionados. As células foram observadas morfologicamente usando um microscópio óptico. Neurônios, vesículas sinápticas e glia foram identificados por β-III-tubulina e MAP-2, Sinapsin-1 e GFAP, respectivamente. Enquanto isso, a imunocitoquímica foi realizada em várias proteínas relacionadas ao neurotransmissor, como acetiltransferase de colina, DYNLL2 e SLC17A9.

No processo de cultura celular primária, as células adquiridas eram redondas com limites brilhantes e claros antes do estado anexo. À medida que os neurônios cresciam, dendritos e axônios começaram a ser distintos. Após 5 a 7 dias de cultura, os neurônios atingiram uma forma madura com projeções longas, que eram ideais para estudos de imagem ou função. Embora a maioria das impurezas e detritos celulares pudessem ser removidos devido à mudança de mídia, certos resíduos ligados ao revestimento de poli-D-lys...

Watch this Scientific Journal Video about Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder at JoVE.com

Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder

Este protocolo tenta estabelecer um protocolo repetível para neurônios primários e isolamento de glia da bexiga de rato para novos experimentos celulares.

Isolamento e Cultura dos Neurônios Primários e Glia da Bexiga Urinária de Ratos Adultos

The lower urinary tract has two main functions, namely, periodic urine storage and micturition; these functions are mediated through central and ...

isolation-culture-primary-neurons-glia-from-adult-rat-urinary

Research

JoVE Journal

Neuroscience

3.8K visualizações.  Guangzhou University of Chinese Medicine. Este protocolo tenta estabelecer um protocolo repetível para neurônios primários e isolamento de glia da bexiga de rato para novos experimentos celulares.

Vídeo: Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and synapse formation and function. An advantage of culturing these neurons is that they readily polarize, forming distinctive axons and dendrites, on a two dimensional substrate at very low densities. This property has made them extremely useful for determining many aspects of neuronal development. Furthermore, by providing glial conditioning for these neurons they will continue to develop, forming functional synaptic connections and surviving for several months in culture. In this protocol we outline a technique to dissect, culture and transfect embryonic mouse hippocampal and cortical neurons. Transfection is accomplished by electroporating DNA into the neurons before plating via nucleofection. This protocol has the advantage of expressing fluorescently-tagged fusion proteins early in development (~4-8hrs after plating) to study the dynamics and function of proteins during polarization, axon outgrowth and branching. We have also discovered that this single transfection before plating maintains fluorescently-tagged fusion protein expression at levels appropriate for imaging throughout the lifetime of the neuron (&gt; 2 months in culture). Thus, this methodology is useful for studying protein localization and function throughout CNS development with little or no disruption of neuronal function.

This protocol outlines the steps required to dissect, transfect via electroporation and culture mouse hippocampal and cortical neurons. Short-term cultures may be used for studies of axon outgrowth and guidance, while long-term cultures can be used for studies of synaptogenesis and dendritic spine analysis.

Nucleofection e Cultura Primária de Neurônios do hipocampo do rato embrionárias e Cortical

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and ...

Neurociência

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.
Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.
A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions1.
At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4, cellular and molecular biology5-8, biochemistry5, imaging and microscopy4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses11, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.
A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously4,12,13. Here we detail a modified protocol suited for cortical neurons. As approximately 20x106 cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.

Here we provide a protocol for culturing rat cortical neurons in the presence of a glial feeder layer. The cultured neurons establish polarity and create synapses, and can be separated from the glia for use in various applications, such as electrophysiology, calcium imaging, cell survival assays, immunocytochemistry, and RNA/DNA/protein isolation.

Bilaminar Co-cultura de neurônios de rato Primária Cortical e Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or ...

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual neurons, researchers are able to analyze properties related to cellular trafficking, cellular structure and individual protein localization using a variety of biochemical techniques. Results from such experiments are critical for testing theories addressing the neural basis of memory and learning. However, unambiguous results from these forms of experiments are predicated on the ability to grow neuronal cultures with minimum contamination by other brain cell types. In this protocol, we use specific media designed for neuron growth and careful dissection of embryonic hippocampal tissue to optimize growth of healthy neurons while minimizing contaminating cell types (i.e. astrocytes). Embryonic mouse hippocampal tissue can be more difficult to isolate than similar rodent tissue due to the size of the sample for dissection. We show detailed dissection techniques of hippocampus from embryonic day 19 (E19) mouse pups. Once hippocampal tissue is isolated, gentle dissociation of neuronal cells is achieved with a dilute concentration of trypsin and mechanical disruption designed to separate cells from connective tissue while providing minimum damage to individual cells. A detailed description of how to prepare pipettes to be used in the disruption is included. Optimal plating densities are provided for immuno-fluorescence protocols to maximize successful cell culture. The protocol provides a fast (approximately 2 hr) and efficient technique for the culture of neuronal cells from mouse hippocampal tissue.

We provide a protocol for the culture of highly purified hippocampal neurons from prenatal mouse brains without the use of a feeder glial cell layer.

Isolamento e Cultura de neurônios do hipocampo de ratos pré-natal

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual ...

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls gastrointestinal motility. A procedure for the isolation and culture of a mixed population of neurons and glia from the myenteric plexus is described. The primary cultures can be maintained for over 7 days, with connections developing among the neurons and glia. The longitudinal muscle strip with the attached myenteric plexus is stripped from the underlying circular muscle of the mouse ileum or colon and subjected to enzymatic digestion. In sterile conditions, the isolated neuronal and glia population are preserved within the pellet following centrifugation and plated on coverslips. Within 24-48 hr, neurite outgrowth occurs and neurons can be identified by pan-neuronal markers. After two days in culture, isolated neurons fire action potentials as observed by patch clamp studies. Furthermore, enteric glia can also be identified by GFAP staining. A network of neurons and glia in close apposition forms within 5 - 7 days. Enteric neurons can be individually and directly studied using methods such as immunohistochemistry, electrophysiology, calcium imaging, and single-cell PCR. Furthermore, this procedure can be performed in genetically modified animals. This methodology is simple to perform and inexpensive. Overall, this protocol exposes the components of the enteric nervous system in an easily manipulated manner so that we may better discover the functionality of the ENS in normal and disease states.

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

We demonstrate a cell culture protocol for the direct study of neuronal and glial components of the enteric nervous system. A neuron/glia mixed culture on coverslips is prepared from the myenteric plexus of adult mouse providing the ability to examine individual neuron and glia function by electrophysiology, immunohistochemical, etc.

Um<em&gt; In-vitro</em&gt; Preparação de neurônios entéricos isolados e células gliais do plexo mioentérico do Rato Adulto

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls ...

Isolation and Culture of Dissociated Sensory Neurons From Chick Embryos

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. Studying neurons in vivo can be challenging due to their complexity, their varied and dynamic environments, and technical limitations. For these reasons, studying neurons in vitro can prove beneficial to unravel the complex mysteries of neurons. The well-defined nature of cell culture models provides detailed control over environmental conditions and variables. Here we describe how to isolate, dissociate, and culture primary neurons from chick embryos. This technique is rapid, inexpensive, and generates robustly growing sensory neurons. The procedure consistently produces cultures that are highly enriched for neurons and has very few non-neuronal cells (less than 5%). Primary neurons do not adhere well to untreated glass or tissue culture plastic, therefore detailed procedures to create two distinct, well-defined laminin-containing substrata for neuronal plating are described. Cultured neurons are highly amenable to multiple cellular and molecular techniques, including co-immunoprecipitation, live cell imagining, RNAi, and immunocytochemistry. Procedures for double immunocytochemistry on these cultured neurons have been optimized and described here.

Cell culture models provide detailed control over environmental conditions and thus provide a powerful platform to elucidate numerous aspects of neuronal cell biology. We describe a rapid, inexpensive, and reliable method to isolate, dissociate, and culture sensory neurons from chick embryos. Details of substrata preparation and immunocytochemistry are also provided.

Isolamento e Cultura de Dissociated neurônios sensoriais de Chick Embriões

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. ...

Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.

The causes of degeneration of midbrain dopaminergic neurons during Parkinson&#8217;s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration.

Isolamento, cultura e Long-Term Manutenção da Primária mesencefálico Dopaminergic neurônios dos cérebros de roedores Embryonic

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes ...

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

Isolamento e Cultura de células estaminais adultas neurais do Subcallosal Zona Rato

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...

Spinal Cord Neurons Isolation and Culture from Neonatal Mice

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on postnatal day 1-3. A mouse litter, usually 4-10 pups born from one breeding pair, is gathered for one experiment, and spinal cords are collected individually from each mouse after euthanasia with isoflurane. The spinal column is dissected out and then the spinal cord is released from the column. The spinal cords are then minced to increase the surface area of delivery for an enzymatic protease that allows for the neurons and other cells to be released from the tissue. Trituration is then used to release the cells into solution. This solution is subsequently fractionated in a density gradient to separate the various cells in solution, allowing for neurons to be isolated. Approximately 1-2.5 x 106 neurons can be isolated from one litter group. The neurons are then seeded onto wells coated with adhesive factors that allow for proper growth and maturation. The neurons take approximately 7 days to reach maturity in the growth and culture medium and can be used thereafter for treatment and analysis.

This study presents a technique for the isolation of neurons from WT neonatal mice. It requires the careful dissection of the spinal cord from the neonatal mouse, followed by the separation of neurons from the spinal cord tissue through mechanical and enzymatic cleavage.

Isolamento e cultura de neurônios da medula espinhal de ratos neonatais

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on ...

Primary Culture of Neurons Isolated from Embryonic Mouse Cerebellum

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model system is to provide a controlled microenvironment and maintain the high survival rate and the natural features of dissociated neuronal and nonneuronal cells as much as possible under in vitro conditions. In this article, we demonstrate a method of isolating primary neurons from the developing mouse cerebellum, placing them in an in vitro environment, establishing their growth, and monitoring their viability and differentiation for several weeks. This method is applicable to embryonic neurons dissociated from cerebellum between embryonic days 12&ndash;18.

Conducting in vitro experiments to reflect in vivo conditions as adequately as possible is not an easy task. The use of primary cell cultures is an important step toward understanding cell biology in a whole organism. The provided protocol outlines how to successfully grow and culture embryonic mouse cerebellar neurons.

Cultura primária de neurônios isolados do cerebelo embrionário do rato

The use of primary cell cultures has become one of the major tools to study the nervous system in vitro. The ultimate goal of using this simplified model ...

Isolation and Culture of Oculomotor, Trochlear, and Spinal Motor Neurons from Prenatal Islmn:GFP Transgenic Mice

Oculomotor neurons (CN3s) and trochlear neurons (CN4s) exhibit remarkable resistance to degenerative motor neuron diseases such as amyotrophic lateral sclerosis (ALS) when compared to spinal motor neurons (SMNs). The ability to isolate and culture primary mouse CN3s, CN4s, and SMNs would provide an approach to study mechanisms underlying this selective vulnerability. To date, most protocols use heterogeneous cell cultures, which can confound the interpretation of experimental outcomes. To minimize the problems associated with mixed-cell populations, pure cultures are indispensable. Here, the first protocol describes in detail how to efficiently purify and cultivate CN3s/CN4s alongside SMNs counterparts from the same embryos using embryonic day 11.5 (E11.5) IslMN:GFP transgenic mouse embryos. The protocol provides details on the tissue dissection and dissociation, FACS-based cell isolation, and in vitro cultivation of cells from CN3/CN4 and SMN nuclei. This protocol adds a novel in vitro CN3/CN4 culture system to existing protocols and simultaneously provides a pure species- and age-matched SMN culture for comparison. Analyses focusing on the morphological, cellular, molecular, and electrophysiological characteristics of motor neurons are feasible in this culture system. This protocol will enable research into the mechanisms that define motor neuron development, selective vulnerability, and disease.

Isolation and Culture of Oculomotor, Trochlear, and Spinal Motor Neurons from Prenatal Islmn:GFP Transgenic Mice

This work presents a protocol to yield homogeneous cell cultures of primary oculomotor, trochlear, and spinal motor neurons. These cultures can be used for comparative analyses of the morphological, cellular, molecular, and electrophysiological characteristics of ocular and spinal motor neurons.

Isolamento e Cultura dos neurônios oculomotores, trochlear e motores espinhais de pré-natal Islmn:GFP Ratos Transgênicos

Oculomotor neurons (CN3s) and trochlear neurons (CN4s) exhibit remarkable resistance to degenerative motor neuron diseases such as amyotrophic lateral ...

Isolamento e Cultura dos Neurônios Primários e Glia da Bexiga Urinária de Ratos Adultos

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