Agradecemos os membros da divisão de ciência do genoma e laboratório de sistemas de biologia e medicina, RCAST, a Universidade de Tóquio. Agradecemos especialmente Dr. H. Aburatani, Dr. T. Kodama, (LSBM, RCAST, a Universidade de Tóquio), Dr. K. Tomizuka, Dr. T. Yoshida e Dr. A. Kunisato (Kyowa Hakko Kirin, Ltd.) para suporte e discussões úteis. Este trabalho foi parcialmente suportado pelo subsídio para jovem cientista (A) (26710005, T.O.), subsídio para a investigação científica em áreas inovadoras (26116711 e 16 H 01567, T.O.) e subsídio para desafiador Pesquisa exploratória (16K 14605, T.O.) do Ministério da educação, cultura, esportes, ciência e tecnologia do Japão, a Fundação de ciência Takeda (T.O.), Fundação de pesquisa do câncer (T.O.) Kobayashi e o projeto para pesquisa do câncer e Evolução terapêutica (P-criar) e a pesquisa de prática de controle do câncer inovadoras da Agência Japonesa para a pesquisa médica e desenvolvimento, AMED (T.O.).

1. preparação do meio de cultura de ácido
<ol><li>
Preparação do controle (pH 7,4) e o meio de cultura de baixo pH (pH 6,8)
	<ol>
<li>Dissolva 4,75 g de pó DMEM sem L-glutamina em 500 mL de água.</li>
		<li>Prepare a solução de 0,33 M NaHCO3 em uma garrafa de pressão-apertado. Atenção: É importante que utilize uma garrafa de pressão apertado NaHCO3 emite CO2 gás quando aquecido e decomposto termicamente.</li>
		<li>Autoclave a médio e a solução. Permitir que esses buffers esfriar a 25 ° C.</li>
		<li>Adicionar 5 mL de 200 mM L-glutamina, 5 mL de penicilina-estreptomicina e 50 mL de soro bovino fetal a 500 mL de DMEM sem bicarbonato.</li>
		<li>Por meio de controle (pH 7,4), adicione 2,4 mL de solução de 0,33 M NaHCO3 por 100 mL de DMEM sem bicarbonato. Por meio do baixo-pH (pH 6,8), adicione 0,6 mL da solução de 0,33 M NaHCO3 por 100 mL de DMEM sem bicarbonato.</li>
		<li>Verificar o pH médio após 24h de incubação a 37 ° C, menos de 5% de CO2. Nota: A quantidade de solução de NaHCO3 precisa ser ajustada dependendo do pH médio, que pode diferir dependendo do teor de CO2 da incubadora e a pressão de ar. Idealmente, pela primeira vez, é melhor medir o pH médio com diferentes concentrações de bicarbonato, para determinar a quantidade adequada de solução de NaHCO3 para adicionar DMEM.<ol>
<li>Recolher o meio de cultura imediatamente após a remoção da placa de cultura a incubadora de CO2 e medir o pH com um medidor de pH. Manter a temperatura média a 37 ° C, utilizando um banho de água se necessário. Medir o pH médio três vezes independentemente. Nota: NaHCO3 solução deve ser armazenada a 4 ° C e deve ser preparadas de cada mídia. Produtos comercialmente disponíveis que podem ser usados neste estudo são listados na Tabela de materiais.</li>
		</ol>
</li>
	</ol>
</li>
	<li>
Preparando o lactato-induzido ou induzido por HCl Ácido meio de cultura
	<ol>
<li>Adicione 74 µ l de solução de lactato ou 125 µ l de 1 M de HCl a 100 mL de meio de controle (preparado na etapa 1.1.5).</li>
		<li>Verificar o pH médio após 24h de incubação a 37 ° C, menos de 5% de CO2. Nota: A quantidade de lactato ou solução de HCl também precisa ser ajustado de acordo com o pH médio, que pode diferir dependendo do teor de CO2 na incubadora e pressão de ar. Idealmente, é melhor medir o pH médio com lactato serial ou concentrações de HCl para determinar a quantidade adequada de ajuste para o pH desejado do meio.</li>
	</ol>
</li>
</ol>2. a colheita e o tratamento de células
<ol><li>Começa a cultivar células a 37 ° C, menos de 5% de CO2 pelo menos 1 semana antes de realizar experimentos. Nota: A linha de celular utilizada neste estudo é listada na Tabela de materiais.</li>
	<li>Células de passagem quando eles alcançam a confluência de 70-90%. Não permita que as células se tornar confluentes. Altere o meio de cada 2-3 dias durante o cultivo diariamente.</li>
	<li>Lave as células pela adição de 5 mL de PBS. Aspirar a PBS e adicionar enzima desmontar como 1ml tripsina.</li>
	<li>Incube a 37 ° C até que as células são arredondadas e começarem a separar.</li>
	<li>Adicionar 5 mL de meio de cultura para as células desanexadas e desactivar a enzima. Transferir a suspensão de células para um tubo de 15 mL e centrifugar por 3 min em 300 x g. Aspire o sobrenadante e ressuspender as células com meio de cultura.</li>
	<li>Conte as células viáveis usando um hemocytometer e azul Trypan.</li>
	<li>Semente 5.0 x 105 células/poço de um prato de 10 cm em 10 mL de meio. Incube as celulas por 24 h a 37 ° C abaixo de 5% de CO2.</li>
	<li>Aspire o meio de cultura, lavam-se as células com 5 mL de PBS e adicionar 10 mL de meio de cultura ácido preparado na secção 1. Incube as celulas por 24 h a 37 ° C abaixo de 5% de CO2.</li>
</ol>3. isolamento de RNA
<ol><li>Aspire o meio de cultura e lavam-se células com 5 mL de PBS. Romper as células, adicionando o reagente de extração de RNA (ver Tabelas de materiais).</li>
	<li>Raspe o prato brevemente, em seguida remover o reagente de extração de RNA com uma pipeta e depositar o RNA extração reagente/lisado celular em um tubo de 1,5 mL. Deixe em temperatura ambiente por 5 min.</li>
	<li>Adicionar 250 clorofórmio µ l e agite o tubo vigorosamente durante cerca de 15 s. deixar em temperatura ambiente por 5 min e centrifugar em ≥ 8, 000 x g durante 5 min.</li>
	<li>Remova cuidadosamente a fase aquosa com uma pipeta. Deixe para trás alguns da fase aquosa. Coloque em outro tubo de 1,5 mL.</li>
	<li>Adicionar a fase aquosa 550 µ l isopropanol e misture delicadamente. Deixe em temperatura ambiente por 5 min. centrifugar a máxima velocidade (≥ 20 000 x g) por 20 min a 4 ° C.</li>
	<li>Despeje o isopropanol e adicionar 1 mL de etanol a 75% em DEPC água tratada. Misture suavemente. Centrifugar a 8.000 x g durante 5 min.</li>
	<li>Pipetar o etanol e deixe que secar ao ar livre as pelotas.</li>
	<li>Adicione que aproximadamente 15-25 µ l (dependendo do rendimento) de DEPC água tratada para a pelota do RNA. Misture bem.</li>
	<li>Medir a concentração de RNA isolado medindo a D.O. a 260 nm, utilizando um espectrofotômetro.</li>
</ol>4. síntese de cDNA
<ol><li>Combinar os seguintes componentes em um tubo de reação de PCR: até 5 µ g RNA total, 1 µ l Oligo dT (50 µM) ou mistura aleatória de Primer (60 µM), 1 µ l 10mm dNTP e água nuclease livre para um volume total de 14 µ l.</li>
	<li>Misture e centrifugar brevemente os componentes utilizando uma centrífuga de bancada pequena (~ 5 s). Desnaturar a amostra de RNA/primer por 5 min a 65 ° C. Girar brevemente (~ 5 s) usando uma pequena bancada centrifugue e colocar a amostra prontamente no gelo pelo menos 1 min.</li>
	<li>Adicione os seguintes componentes: 4 µ l 5 x buffer de transcrição reversa, 1 µ l Transcriptase reversa (200 U / µ l), 1 µ l inibidor de RNase (40 U / µ l). Nota: Exemplos de kits comercialmente disponíveis são mostrados na Tabela de materiais
</li>
	<li>Tampa do tubo, misture e brevemente centrifugar o conteúdo utilizando uma centrífuga de bancada pequena (~ 5 s). Incube a mistura de reação combinada a 50-55 ° C por 10 min.</li>
	<li>Inativar a reação incubando a 80 ° C por 10 min.</li>
</ol>5. medida da expressão do Gene ácido-responsivo usando PCR em tempo real
<ol><li>Combinar os seguintes componentes em uma placa de reação 384-bem: 1 µ l modelo do cDNA, 0,75 µ l 10 X N',N'-dimethyl-N-[4-[(E)-(3-methyl-1,3-benzothiazol-2-ylidene)methyl]-1-phenylquinolin-1-ium-2-yl]-N-propylpropane-1,3-diamine, 0,3 µ l 50 µM para a frente primeira demão, 0,3 µ l 50 µM reverso da primeira demão, polymerase de Taq 1 µ l, 2,5 µ l 10 x buffer do polymerase de Taq, 2,5 µ l 20 mM dNTP, 16.65 µ l água. Nota: O exemplo dos kits comercialmente disponíveis são mostrados na Tabela de materiais. Uma lista das primeiras demão é mostrada na tabela 1. Uma placa de 96 poços de reação também pode ser usada nesta etapa. Outras sondas (por exemplo, sonda Taqman) também podem ser usadas.</li>
	<li>Configurar a experiência e o seguinte programa PCR em um sistema de PCR em tempo real: 50 ° C por 2 min, 1 ciclo; 95 ° C por 10 min, 1 ciclo; 95 ° C por 15 s e 60 ° C por 1 min, 40 ciclos; 95 ° C por 15 s, 1 ciclo; 60 ° C durante 15 s, 1 ciclo; 95 ° C por 15 s, 1 ciclo.</li>
	<li>Coloque a placa no instrumento qPCR. Inicie o programa. Nota: Upregulation dos genes responsivos pH MSMO1, INSIG1e IDI1 podem ser medido pelos níveis de proteína.</li>
</ol>

<ol>
	<li>Cairns, R. A., Harris, I. S., Mak, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+cancer+cell+metabolism.">Regulation of cancer cell metabolism.</a> Nature reviews. Cancer. 11 (2), 85-95 (2011).</li><li>Chang, C. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+Competition+in+the+Tumor+Microenvironment+Is+a+Driver+of+Cancer+Progression.">Metabolic Competition in the Tumor Microenvironment Is a Driver of Cancer Progression.</a> Cell. 162 (6), 1229-1241 (2015).</li><li>Hsu, P. P., Sabatini, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+cell+metabolism:+Warburg+and+beyond.">Cancer cell metabolism: Warburg and beyond.</a> Cell. 134 (5), 703-707 (2008).</li><li>Gerweck, L. E., Seetharaman, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+pH+gradient+in+tumor+versus+normal+tissue:+potential+exploitation+for+the+treatment+of+cancer.">Cellular pH gradient in tumor versus normal tissue: potential exploitation for the treatment of cancer.</a> Cancer research. 56 (6), 1194-1198 (1996).</li><li>Webb, B. A., Chimenti, M., Jacobson, M. P., Barber, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dysregulated+pH:+a+perfect+storm+for+cancer+progression.">Dysregulated pH: a perfect storm for cancer progression.</a> Nature Rev. Cancer. 11 (9), 671-677 (2011).</li><li>Gatenby, R. A., Gillies, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+do+cancers+have+high+aerobic+glycolysis?.">Why do cancers have high aerobic glycolysis?.</a> Nature reviews. Cancer. 4 (11), 891-899 (2004).</li><li>McBrian, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+acetylation+regulates+intracellular+pH.">Histone acetylation regulates intracellular pH.</a> Mol Cell. 49 (2), 310-321 (2013).</li><li>Damaghi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+acidosis+in+the+tumour+microenvironment+selects+for+overexpression+of+LAMP2+in+the+plasma+membrane.">Chronic acidosis in the tumour microenvironment selects for overexpression of LAMP2 in the plasma membrane.</a> Nat Commun. 6, 8752 (2015).</li><li>Corbet, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acidosis+Drives+the+Reprogramming+of+Fatty+Acid+Metabolism+in+Cancer+Cells+through+Changes+in+Mitochondrial+and+Histone+Acetylation.">Acidosis Drives the Reprogramming of Fatty Acid Metabolism in Cancer Cells through Changes in Mitochondrial and Histone Acetylation.</a> Cell Metab. 24 (2), 311-323 (2016).</li><li>Chen, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genomic+analysis+of+lactic+acidosis+and+acidosis+response+in+human+cancers.">The genomic analysis of lactic acidosis and acidosis response in human cancers.</a> PLoS genetics. 4 (12), e1000293 (2008).</li><li>Kondo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+Acidic+pH+Activates+the+Sterol+Regulatory+Element-Binding+Protein+2+to+Promote+Tumor+Progression.">Extracellular Acidic pH Activates the Sterol Regulatory Element-Binding Protein 2 to Promote Tumor Progression.</a> Cell Rep. 18 (9), 2228-2242 (2017).</li></ol>

O autor Ayano Kondo é um empregado da Kyowa Hakko Kirin co., Ltd.

Aqui, descrevemos um sistema de cultura simples pH ácido e seu processo de avaliação. A combinação de três métodos de acidificação média, ou seja, concentração de bicarbonato reduzido, adição de lactato e adição de HCl, permitiu-nos investigar o mecanismo de pH-resposta completamente e comparar a resposta celular a outro tumor Microenvironmental condições tais como a fome de hipóxia ou nutriente.
A chave deste método é para determinar as concentrações adequadas ...

O microambiente do tumor tem um papel crítico na progressão do tumor e câncer célula metabolismo1,2,3. As células cancerosas são frequentemente expostas a condições tais como hipóxia, privação de nutrientes e ácido pH extracelular (pHe). No entanto, o papel de pHe em progressão do tumor não foi clarificado tão extensivamente como fome de hipóxia ou nutriente. O pHe no tecido do tumor pode se tornar ácida, atingindo aproximadamente pH 6,84,5. O pH ácido surge da excreção glicolítico aeróbia e anaeróbica de prótons (H+) e lactato pela proliferação do câncer células5,6.
Estudos recentes revelaram que ácida induzida pela pHe histona deacetilação, oxidação do ácido graxo e exocitose de lisossomos ácidos para adaptação ao ambiente ácido grave7,8,9,10. No entanto, os mecanismos através do qual acidificação do extracelular afeta o comportamento do câncer e a identidade da chave reguladores no microambiente do tumor de pH ácido não foram totalmente determinados. Além disso, vários relatos descritos vários meios de pH ácido, usando pouco claras concentrações de bicarbonato, tampão Tris, tubulações e HEPES ou lactato e HCl, mas há poucos relatórios para demonstrar a estabilidade do ajustado média nem abrangente comparação de vários meios distintos de cultura ácida7,8,9,10
Para elucidar os reguladores chaves e alterações metabólicas em células cancerosas no contexto da acidificação do extracelular, que estabeleceu um modelo de cultura simples em vitro para manter um pHe ácida e examinou o papel de pHe em células de câncer11. Usando esse método, mantivemos um meio de cultura ácido com um pHe de 6,8 a 37 ° C abaixo dos 5% CO2, com as concentrações de bicarbonato reduzida do meio de cultura. pH 7,4 utilizou-se como normal e controle médio. O pH médio foi mantido pelo menos 24 h e gradualmente diminuiu 72 h durante a cultura de células de câncer pancreático PANC-1 e 1-AsPC. Porque reforçada glicólise acelera a excreção de prótons, não só mas também lactato7,8,9, estabelecemos também um método de cultura imitando acidose induzida por lactato por adição de lactato, em vez de reduzir o concentração de bicarbonato. Além disso, induzida por HCl Ácido pHe no meio nos permite excluir a possibilidade de respostas celulares ao meio de cultura de pH ácido não são devido a uma concentração reduzida de bicarbonato. Além disso, usando vários meios de comunicação com um pH de 6,4 a 7,4 com concentração diferente de bicarbonato, podemos avaliar a extensão dos efeitos de pHe em respostas celulares.

<table>
	<tbody>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium&#8220;Nissui&#8221; 2</td>
			<td>Nissui</td>
			<td>05919</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Thermo Fisher&#160;</td>
			<td>10438</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Wako</td>
			<td>191-01305</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine solution</td>
			<td>Thermo Fisher&#160;</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin Mixed Solution(Stabilized)</td>
			<td>Nacalai</td>
			<td>09367-34</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5g/l-Tripsin/0.53mmol/l-EDTA Solution, with Phenol Red</td>
			<td>Nacalai</td>
			<td>32778-05</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5%-Trypan Blue Stain Solution</td>
			<td>Nacalai</td>
			<td>29853-34</td>
			<td></td>
		</tr>
		<tr>
			<td>Lactic acid solution</td>
			<td>Sigma-Aldrich</td>
			<td>252476</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid solution</td>
			<td>Sigma-Aldrich</td>
			<td>H9892</td>
			<td></td>
		</tr>
		<tr>
			<td>RNeasy Plus Mini Kit</td>
			<td>QIAGEN</td>
			<td>74134</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperScript IV First-Strand Synthesis System</td>
			<td>Thermo Fisher&#160;</td>
			<td>18091</td>
			<td></td>
		</tr>
		<tr>
			<td>Power SYBR Green PCR Master Mix&#160;</td>
			<td>Applied Biosystems&#160;</td>
			<td>4368702&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Cell lines</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PANC-1</td>
			<td>ATCC</td>
			<td>CRL-1469</td>
			<td></td>
		</tr>
		<tr>
			<td>AsPC-1</td>
			<td>ATCC</td>
			<td>CRL-1682</td>
			<td></td>
		</tr>
		<tr>
			<td>KMS-6</td>
			<td>JCRB Cell Bank</td>
			<td>JCRB0432</td>
			<td></td>
		</tr>
		<tr>
			<td>TIG</td>
			<td>JCRB Cell Bank</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MRC9</td>
			<td>ATCC</td>
			<td>CCL-212</td>
			<td></td>
		</tr>
		<tr>
			<td>HUVEC</td>
			<td>ATCC</td>
			<td>CRL-1730</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop One Microvolume UV-Vis Spectrophotometer with Wi-Fi</td>
			<td>Thermo Fisher</td>
			<td>ND-ONE-W</td>
			<td></td>
		</tr>
		<tr>
			<td>QuantStudio 5 Real-Time PCR System</td>
			<td>Thermo Fisher</td>
			<td>CRL-1682</td>
			<td></td>
		</tr>
	</tbody>
</table>

establishment of an extracellular acidic ph culture system

Condições do microambiente do tumor, como hipóxia ou inanição nutriente, desempenham um papel crítico na progressão do câncer e malignidade. No entanto, o papel do pH extracelular ácido na agressividade do tumor e seu mecanismo subjacente não foi extensivamente estudado em comparação com as condições de inanição hipóxica ou nutrientes. Além disso, um método de cultura bem definidos para imitar o microambiente do tumor extracelular ácidas não totalmente relatou.
Aqui nós apresentamos um método de cultura simples em vitro para manter o pH extracelular ácido usando reduzido bicarbonato e lactato aumentado ou concentrações de HCl em meio de cultura. O pH médio foi mantido pelo menos 24 h e gradualmente diminuiu de 72 h após a cultura de células de câncer pancreático PANC-1 e 1-AsPC. Três condições distintas mídia ácida neste estudo altamente upregulated pH-responsivo genes tais como MSMO1, INSIG1e IDI1 em comparação com hipoxia ou inanição nutriente. O upregulation destes genes pode ser usado como um marcador de pH ácido. Estas técnicas simples são benéficas para elucidar os mecanismos subjacentes de malignidade do tumor sob o microambiente do tumor ácidas. Portanto, nosso sistema de cultura de pH ácido extracelular permite a descoberta de respostas celulares pH ácido não só nas células cancerosas, mas também em células primárias, tais como células tubulares renais, em relação as outros ácidos distúrbios incluindo, cetoacidose diabética, acidose láctica, acidose tubular renal e acidose respiratória.

Para determinar a concentração de bicarbonato apropriado, estamos preparados DMEM no intervalo de 0 - 8 mM NaHCO3 (concentração final do meio de cultura) e teve sucesso na preparação de meios de cultura com pH variando de 6,4-7,4 (Figura 1). Estamos preparados DMEM com 8mm NaHCO3 (pH 7,4) como um meio de controle e 2mm NaHCO3 (pH 6,8) como um meio com pH ácido de acordo com um relatório anterior, afirmando que o pH ext...

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Establishment of an Extracellular Acidic pH Culture System

O microambiente do tumor ácido tem um papel crítico na progressão do tumor. Para avaliar os efeitos do pH extracelular ácido sobre câncer células in vitro, estabelecemos sistemas simples cultura ácidas.

Estabelecimento de um pH extracelular ácido sistema de cultura

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the ...

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14.5K visualizações.  Kyowa Hakko Kirin Co., Ltd. O microambiente do tumor ácido tem um papel crítico na progressão do tumor. Para avaliar os efeitos do pH extracelular ácido sobre câncer células in vitro, estabelecemos sistemas simples cultura ácidas.

Vídeo: Establishment of an Extracellular Acidic pH Culture System

Measuring Plant Cell Wall Extension (Creep) Induced by Acidic pH and by Alpha-Expansin

Growing plant cell walls characteristically exhibit a property known as 'acid growth', by which we mean they are more extensible at low pH (&lt; 5) 1. The plant hormone auxin rapidly stimulates cell elongation in young stems and similar tissues at least in part by an acid-growth mechanism 2, 3. Auxin activates a H+ pump in the plasma membrane, causing acidification of the cell wall solution. Wall acidification activates expansins, which are endogenous cell wall-loosening proteins 4, causing the cell wall to yield to the wall tensions created by cell turgor pressure. As a result, the cell begins to enlarge rapidly. This 'acid growth' phenomenon is readily measured in isolated (nonliving) cell wall specimens. The ability of cell walls to undergo acid-induced extension is not simply the result of the structural arrangement of the cell wall polysaccharides (e.g. pectins), but depends on the activity of expansins 5. Expansins do not have any known enzymatic activity and the only way to assay for expansin activity is to measure their induction of cell wall extension. This video report details the sources and preparation techniques for obtaining suitable wall materials for expansin assays and goes on to show acid-induced extension and expansin-induced extension of wall samples prepared from growing cucumber hypocotyls.
 To obtain suitable cell wall samples, cucumber seedlings are grown in the dark, the hypocotyls are cut and frozen at -80 &deg;C. Frozen hypocotyls are abraded, flattened, and then clamped at constant tension in a special cuvette for extensometer measurements. To measure acid-induced extension, the walls are initially buffered at neutral pH, resulting in low activity of expansins that are components of the native cell walls. Upon buffer exchange to acidic pH, expansins are activated and the cell walls extend rapidly. We also demonstrate expansin activity in a reconstitution assay. For this part, we use a brief heat treatment to denature the native expansins in the cell wall samples. These inactivated cell walls do not extend even in acidic buffer, but addition of expansins to the cell walls rapidly restores their ability to extend.

Measuring Plant Cell Wall Extension Creep Induced by Acidic pH and by Alpha-Expansin

We demonstrate the use of a constant-force extensometer to measure long-term extension (creep) of plant cell wall specimens induced by acidic buffers and expansin protein.

Medição Usina Extensão parede celular (Creep) induzida pelo pH ácido e por Alpha-Expansin

Growing plant cell walls characteristically exhibit a property known as 'acid growth', by which we mean they are more extensible at low pH (&lt; 5) 1. The ...

Biologia

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for discovering new therapeutic strategies. Advancements in imaging technology have enabled quantitative investigation of the organization and maturing of the body plan, but later stage embryonic morphogenesis is less clear. Chicken embryos are an attractive vertebrate animal model system for this application because of its ease of culture and surgical manipulation. Early embryos can be cultured for a short time on filter paper rings, which enables complete optical access for cell patterning and fate studies1,2. Studying advanced developmental processes such as cardiac morphogenesis are traditionally performed through a window of the eggshell3-5, but this technique limits optical access due to window size. We previously developed a simple method to culture whole embryos ex-ovo on hexagonal weigh boats for up to 10 days, which enabled high resolution imaging via ultrasonography6,7. These cultures were difficult to transport, limiting the types of imaging tools available for live experiments. We here present an improved shell-less culture system with a cost-effective, portable environmental chamber. Eggs were cracked onto a hammock created by a polyurethane membrane (cling wrap) affixed circumferentially to a plastic cup partially filled with sterile water. The dimensions of the circumference and depth of the hammock were both critical to maintain surface tension, while the mechanics of the hammock and water beneath helped dampen vibrations induced by transportation. A small footprint circulating water bath was also developed to enable continuous temperature control during experimentation. We demonstrate the ability to culture embryos in this way for at least 14 days without morphogenic defect or delay and employ this system in several microsurgical and imaging applications.

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

In this article, we present a simple methodology to enable long-term ex-ovo avian embryo culture. This technique is ideal for longitudinal experimentation requiring complete optical accessibility and/or sterile transportation in avian embryos.

Um Ex-ovo Sistema de Cultura de embriões de frango Indicado para aplicações de imagem e Microcirurgia

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for ...

The Method of Rodent Whole Embryo Culture using the Rotator-type Bottle Culture System

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development and growth of mammalian embryos are critically dependent on the function of the placenta, WEC technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages during embryonic day (E) 6.5-E12.5 in the mouse or E8.5-E14.5 in the rat 2, 3, 4. In WEC, we can directly target desired areas of embryos using fine glass capillaries because embryos can be manipulated under the microscope. Therefore, rodent WEC is very useful technique when we want to study dynamic developmental processes of postimplanted mammalian embryos. Up to date, several types of WEC systems have been developed 1. Among those, the rotator-type bottle culture system is most popular and suitable for long-term culture of embryos at midgestation, i.e., after E9.5 and E11.5 in the mouse and rat, respectively 1. In this video protocol, we demonstrate our standard procedures of rat WEC after E12.5 using a refined model of the original rotator system, which was designed by New and Cockroft 5, 6, and introduce various applications of WEC technique for studies in mammalian developmental biology.

Whole embryo culture technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages. In this video protocol, we demonstrate our standard procedures of rat whole embryo culture after E12.5 using the rotator-type bottle culture system.

O método de cultura de embriões de roedores inteiro usando o Rotator do tipo Sistema de Cultura Garrafa

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development ...

An Ex vivo Culture System to Study Thyroid Development

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are produced by differentiated thyrocytes, organized in closed spheres called follicles, while calcitonin is synthesized by C-cells, interspersed in between the follicles and a dense network of blood capillaries. Although adult thyroid architecture and functions have been extensively described and studied, the formation of the &#8220;angio-follicular&#8221; units, the distribution of C-cells in the parenchyma and the paracrine communications between epithelial and endothelial cells is far from being understood.
This method describes the sequential steps of mouse embryonic thyroid anlagen dissection and its culture on semiporous filters or on microscopy plastic slides. Within a period of four days, this culture system faithfully recapitulates in vivo thyroid development. Indeed, (i) bilobation of the organ occurs (for e12.5 explants), (ii) thyrocytes precursors organize into follicles and polarize, (iii) thyrocytes and C-cells differentiate, and (iv) endothelial cells present in the microdissected tissue proliferate, migrate into the thyroid lobes, and closely associate with the epithelial cells, as they do in vivo.
Thyroid tissues can be obtained from wild type, knockout or fluorescent transgenic embryos. Moreover, explants culture can be manipulated by addition of inhibitors, blocking antibodies, growth factors, or even cells or conditioned medium. Ex vivo development can be analyzed in real-time, or at any time of the culture by immunostaining and RT-qPCR.
In conclusion, thyroid explant culture combined with downstream whole-mount or on sections imaging and gene expression profiling provides a powerful system for manipulating and studying morphogenetic and differentiation events of thyroid organogenesis.

An Ex vivo Culture System to Study Thyroid Development

This protocol describes dissection of mouse embryonic thyroid anlagen and the culture of explants on semiporous filters or on microscopy plastic slides. This system is ideal to study morphogenetic or differentiation events occurring during thyroid development of wild type or knockout embryos, and is amenable to gain- and loss-of-function experiments.

Uma<em&gt; Ex vivo</em&gt; Sistema Cultura para estudar o desenvolvimento da tireóide

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are ...

Development of an Insert Co-culture System of Two Cellular Types in the Absence of Cell-Cell Contact

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt to make straightforward the very complex relationships between the several cellular subtypes that compose multicellular organisms, in vitro techniques have been developed to help researchers acquire a detailed understanding of single cell populations. One of these techniques uses inserts with a permeable membrane allowing secreted soluble factors to diffuse. Thus, a population of cells grown in inserts can be co-cultured in a well or dish containing a different cell type for evaluating cellular changes following paracrine signaling in the absence of cell-cell contact. Such insert co-culture systems offer various advantages over other co-culture techniques, namely bidirectional signaling, conserved cell polarity and population-specific detection of cellular changes. In addition to being utilized in the field of inflammation, cancer, angiogenesis and differentiation, these co-culture systems are of prime importance in the study of the intricate relationships that exist between the different cellular subtypes present in the central nervous system, particularly in the context of neuroinflammation. This article offers general methodological guidelines in order to set up an experiment in order to evaluating cellular changes mediated by secreted soluble factors using an insert co-culture system. Moreover, a specific protocol to measure the neuroinflammatory effects of cytokines secreted by lipopolysaccharide-activated N9 microglia on neuronal PC12 cells will be detailed, offering a concrete understanding of insert co-culture methodology.

In multicellular organisms, secreted soluble factors elicit responses from different cell types as a result of paracrine signaling. Insert co-culture systems offer a simple way to assess the changes mediated by secreted soluble factors in the absence of cell-cell contact.

Desenvolvimento de um Sistema Insert Co-cultura de dois tipos celulares na ausência de célula-célula Contact

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt ...

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH (pHi) is influenced by many ion transporters and thus monitoring pHi is a useful tool for assessing transporter activity. Modern Genetically Encoded pH-Indicators (GEpHIs) provide optical quantification of pHi in intact cells on a cellular and subcellular scale. This protocol describes real-time quantification of cellular pHi regulation in Malpighian Tubules (MTs) of Drosophila melanogaster through ex vivo live-imaging of pHerry, a pseudo-ratiometric GEpHI with a pKa well-suited to track pH changes in the cytosol. Extracted adult fly MTs are composed of morphologically and functionally distinct sections of single-cell layer epithelia, and can serve as an accessible and genetically tractable model for investigation of epithelial transport. GEpHIs offer several advantages over conventional pH-sensitive fluorescent dyes and ion-selective electrodes. GEpHIs can label distinct cell populations provided appropriate promoter elements are available. This labeling is particularly useful in ex vivo, in vivo, and in situ preparations, which are inherently heterogeneous. GEpHIs also permit quantification of pHi in intact tissues over time without need for repeated dye treatment or tissue externalization. The primary drawback of current GEpHIs is the tendency to aggregate in cytosolic inclusions in response to tissue damage and construct over-expression. These shortcomings, their solutions, and the inherent advantages of GEpHIs are demonstrated in this protocol through assessment of basolateral proton (H+) transport in functionally distinct principal and stellate cells of extracted fly MTs. The techniques and analysis described are readily adaptable to a wide variety of vertebrate and invertebrate preparations, and the sophistication of the assay can be scaled from teaching labs to intricate determination of ion flux via specific transporters.

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

Cellular ion transport can often be assessed by monitoring intracellular pH (pHi). Genetically Encoded pH-Indicators (GEpHIs)&#160;provide optical quantification of intracellular pH in intact cells. This protocol details the quantification of intracellular pH through cellular ex vivo live-imaging of Malpighian tubules of Drosophila melanogaster with pHerry, a pseudo-ratiometric genetically encoded pH-indicator.

PH intracelular de quantificação de óptica em Drosophila melanogaster túbulo de Malpighi epitélios com um indicador de pH fluorescente geneticamente codificado

Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH ...

Establishment of a Clonal Culture of Unicellular Conjugating Algae

It is essential to establish clonal cultures of microalgae for use in studies of various topics, such as physiology, genetics, taxonomy, and microbiology. Thus, it is extremely important to develop techniques to establish clonal cultures. In this article, we demonstrate the establishment of clonal cultures of a conjugating alga. Water samples are collected from the field. Subsequently, cells are isolated using a glass capillary pipette, placed in media, and grown under conditions suitable for generating a clonal culture.

In this article, we demonstrate the establishment of clonal cultures of unicellular conjugating algal species collected from a natural field site.

Estabelecimento de uma cultura Clonal de algas unicelulares de conjugação

It is essential to establish clonal cultures of microalgae for use in studies of various topics, such as physiology, genetics, taxonomy, and microbiology. ...

Measuring the Confluence of iPSCs Using an Automated Imaging System

This study focuses on understanding how growing iPSCs on different ECM coating substrates can affect cell confluence. A protocol to assess iPSC confluence in real time has been established without the need to count cells in single cell suspension to avoid any growth perturbation. A high-content image analysis system was used to assess iPCS confluence on 4 different ECMs over time in an automated manner. Different analysis settings were used to assess cell confluence of adherent iPSCs and only a slight difference (at 24 and 48 hours with laminin) has been observed whether a 60, 80 or 100% mask was applied. We also show that laminin lead to the best confluence compared to Matrigel, vitronectin and fibronectin.

The goal of the protocol is to compare different extracellular matrix (ECM) coating conditions to assess how differential coating affects the growth rate of induced pluripotent stem cells (iPSCs). In particular, we aim to set up conditions to obtain optimal growth of iPSC cultures.

Medindo a confluência de iPSCs usando um sistema automatizado de imagem.

This study focuses on understanding how growing iPSCs on different ECM coating substrates can affect cell confluence. A protocol to assess iPSC confluence ...

Culture of Piglet Intestinal 3D Organoids from Cryopreserved Epithelial Crypts and Establishment of Cell Monolayers

Intestinal organoids are increasingly being used to study the gut epithelium for digestive disease modeling, or to investigate interactions with drugs, nutrients, metabolites, pathogens, and the microbiota. Methods to culture intestinal organoids are now available for multiple species, including pigs, which is a species of major interest both as a farm animal and as a translational model for humans, for example, to study zoonotic diseases. Here, we give an in-depth description of a procedure used to culture pig intestinal 3D organoids from frozen epithelial crypts. The protocol describes how to cryopreserve epithelial crypts from the pig intestine and the subsequent procedures to culture 3D intestinal organoids. The main advantages of this method are (i) the temporal dissociation of the isolation of crypts from the culture of 3D organoids, (ii) the preparation of large stocks of cryopreserved crypts derived from multiple intestinal segments and from several animals at once, and thus (iii) the reduction in the need to sample fresh tissues from living animals. We also detail a protocol to establish cell monolayers derived from 3D organoids to allow access to the apical side of epithelial cells, which is the site of interactions with nutrients, microbes, or drugs. Overall, the protocols described here is a useful resource for studying the pig intestinal epithelium in veterinary and biomedical research.

Here, we describe a protocol to culture pig intestinal 3D organoids from cryopreserved epithelial crypts. We also describe a method to establish cell monolayers derived from 3D organoids, allowing access to the apical side of epithelial cells.

Cultura de Organoides 3D Intestinais de Leitões a partir de Criptas Epiteliais Criopreservadas e Estabelecimento de Monocamadas Celulares

Intestinal organoids are increasingly being used to study the gut epithelium for digestive disease modeling, or to investigate interactions with drugs, ...

Isolamento Aprimorado de Vesículas Extracelulares Bacterianas para Microbiota Multi-Ômica

Isolation and Purification of Bacterial Extracellular Vesicles from Human Feces Using Density Gradient Centrifugation

Bacterial extracellular vesicles (BEVs) are nanovesicles derived from bacteria that play an active role in bacteria-bacteria and bacteria-host communication, transferring bioactive molecules such as proteins, lipids, and nucleic acids inherited from the parent bacteria. BEVs derived from the gut microbiota have effects within the gastrointestinal tract and can reach distant organs, resulting in significant implications for physiology and pathology. Theoretical investigations that explore the types, quantities, and roles of BEVs derived from human feces are crucial for understanding the secretion and function of BEVs from the gut microbiota. These investigations also necessitate an improvement in the current strategy for isolating and purifying BEVs.
This study optimized the isolation and purification process of BEVs by establishing two density gradient centrifugation (DGC) modes: Top-down and Bottom-up. The enriched distribution of BEVs was determined in fractions 6 to 8 (F6-F8). The effectiveness of the approach was evaluated based on particle morphology, size, concentration, and protein content. The particle and protein recovery rates were calculated, and the presence of specific markers was analyzed to compare the recovery and purity of the two DGC modes. The results indicated that the Top-down centrifugation mode had lower contamination levels and achieved a recovery rate and purity similar to that of the Bottom-up mode. A centrifugation time of 7 h was sufficient to achieve a fecal BEV concentration of 108/mg.
Apart from feces, this method could be applied to other body fluid types with proper modification according to the differences in components and viscosity. In conclusion, this detailed and reliable protocol would facilitate the standardized isolation and purification of BEVs and thus, lay a foundation for subsequent multi-omics analysis and functional experiments.

Autor em destaque: acelerando a pesquisa sobre separação e heterogeneidade de vesículas extracelulares bacterianas 

This study describes a method to isolate and purify bacterial extracellular vesicles (BEVs) enriched from human feces via density gradient centrifugation (DGC), identifies the physical characteristics of BEVs from morphology, particle size, and concentration, and discusses the potential applications of the DGC approach in clinical and scientific research.