This work was supported by the Seed Grant Award from California Northstate University College of Pharmacy (to AMN). The authors would like to thank California Northstate University College of Pharmacy for covering the publication cost of this article, and George Talbott for his help in editing this manuscript.

1. A manutenção das células Caco-2 <ol><li> Usando regulares pratos de cultura de tecidos <ol><li> Descongelar as células Caco-2 a partir de um frasco congelado colocando o frasco num banho de água a 37 C °, e adicioná-los imediatamente para uma placa de 10 cm de cultura de tecidos contendo 10 ml de meio de crescimento (15% de soro bovino fetal pré-aquecido (FBS ) em Dulbecco Modified Eagle Médium (DMEM)). <ol><li> Quando as células Caco-2 atingiram 50-70% de confluência, dividi-los 1: 6 por incubação das células com 3 ml de 0,05% de tripsina / 0,53 mM de ácido etilenodiaminotetracético (EDTA) a 37 ° C até que eles são destacados (15 min) . Para evitar aglomeração de células, mistura as células várias vezes por pipetagem suave. Adicionar 0,5 ml das células tripsinizadas a uma placa de 10 cm de cultura de tecidos contendo 10 ml de meio de crescimento pré-aquecido. </li></ol></li><li> Agite ligeiramente os pratos várias vezes para a frente e para trás seguido por uma direção para a esquerda e para a direita. Evite rodar os pratosuma vez que pode resultar numa dispersão celular desigual. </li><li> Colocar as cápsulas sobre uma superfície plana em uma incubadora a 37 ° C fornecido com 5% de CO 2. Superfícies inclinadas também podem resultar numa dispersão celular desigual. </li><li> Troque a mídia de crescimento de um dia após o início da incubação. </li><li> Monitorar as células e gravar no dia em que chegar a confluência. Isso levará cerca de uma semana para as células para alcançar confluência do dia eles estão divididos. </li><li> Mudar o meio de crescimento, duas vezes por semana antes de as células atingirem confluência. Uma vez que as células atingiram a confluência, muda os meios de crescimento a cada dois dias. Depois que as células são 7-dias pós-confluentes, alterar a mídia de crescimento diário. Nota: As células estão prontas para experiências quando eles são de 13 dias pós-confluentes (Figura 1). Uma vez que as células acabará por se tornar menos eficaz na produção de lipoproteínas, utilização de células que se encontram entre 13 a 17 dias pós-confluentes 8. Vá para a etapa 3 sobre como conduzir o experimento.</li></ol></li><li> Usando o sistema de membrana permeável <ol><li> Semente as células Caco-2 no inserto membrana permeável tal como descrito no passo 1.1.1.1. acima. Evite o uso de células a partir de um frasco congelado porque o seu crescimento celular é normalmente mais lenta (período de recuperação). Resumidamente, adicionar 0,5 ml das células tripsinizadas para a câmara de apical (compartimento superior) que contém 10 ml de meio de crescimento pré-aquecido. Além disso, adicionar 10 ml de meio de crescimento pré-aquecido para a câmara basolateral (compartimento inferior). Nota: Para as melhores práticas, adicione os meios de crescimento para a câmara apical primeiro e depois para a câmara basolateral. Ao remover a velha mídia, remova a mídia basolateral perante a mídia apical. Evitar picar a membrana de policarbonato, uma vez que pode romper a membrana. </li><li> Devido à visibilidade pobre célula através da membrana de policarbonato, semear as células Caco-2 em um prato de cultura de tecidos regular com densidade semelhante, e usar este prato para julgar a confluência celular. </li><li> Siga sPTE 1.1.2-1.1.6 acima. </li></ol></li></ol> 2. Gene Overexpression <ol><li> Usando a abordagem de transfecção regulares <ol><li> Semente as células Caco-2 em um prato de cultura de tecidos como descrito no 1.1.1.1.-1.1.4 supra. Nota: Quando as células são cerca de 40-50% confluentes, eles estão prontos para ser transfectada. </li><li> Adicionar 67 ul de reagente de transfecção de 472 ul de meio de soro reduzido os num tubo de microcentrífuga. Evitar o contacto do reagente de transfecção não diluída com a parede do tubo. </li><li> Adicionar 23 g de pLL3.7 reforçada proteína verde fluorescente (EGFP) 9 para o reagente de transfecção / meio de soro reduzido mistura. </li><li> Incubar a mistura durante 20 min à temperatura ambiente. </li><li> Substituir o meio de crescimento das células Caco-2 &#39;com 8 ml de 10% de FBS em meio DMEM. </li><li> Adicionar a mistura de ADN / reagente de transfecção / meio de soro reduzido, gota a gota para o prato. </li><li> Agite ligeiramente os pratos várias vezes em uma frente e badirecção ckward seguido por um sentido para a esquerda e para a direita. </li><li> Coloque o prato no 37 ° C incubadora. </li><li> Substitua a mídia no prato com 10 ml de meio de crescimento por dia após o início da incubação. </li><li> Monitorar a expressão do gene. <ol><li> Monitorar a expressão de genes sem 4 &#39;, 6-diamidino-2-fenilindole (DAPI) coloração. Usando um microscópio de fluorescência, determinar a percentagem de células que são verdes. A percentagem representa a eficiência da transfecção. </li><li> Monitorar a expressão gênica com coloração DAPI. <ol><li> Lavam-se as células com solução salina tamponada com fosfato (PBS) três vezes. </li><li> Fixar as células por adição de 10 ml de formaldeído a 4% em PBS (10 minutos de incubação à TA). </li><li> Lavam-se as células com PBS três vezes. </li><li> Incubar as células no escuro durante 15 min à temperatura ambiente com 8 ml de PBS contendo 1: 500 DAPI, 1% de BSA, 0,01% de digitonina. </li><li> Lavam-se as células com PBS três vezes. </li><li> VCcante um microscópio de fluorescência, determinar o número de células que são verde / azul em relação àqueles que são azuis (Figura 2 painel superior). A percentagem calculada representa a eficiência da transfecção. </li></ol></li></ol></li></ol></li><li> Usando o sistema de sobre-expressão de lentivírus 10 <ol><li> Rim embrionário (HEK) Semear as células em um humanos 293T 15 centímetros prato de cultura de tecidos (10% de FBS em DMEM como seu meio de crescimento). Nota: Quando as células são cerca de 60-70% confluentes, eles estão prontos para ser transfectada. </li><li> Adicionar 162 ul de reagente de transfecção de 1133 ul de os meio de soro reduzido em um tubo de microcentrífuga de 11. Evitar o contacto do reagente de transfecção não diluída com a parede do tubo. </li><li> Adicionar 24 g de pLL3.7 eGFP (ou outra construção), 15,6? G de elemento de resposta a Rev (RRE), 6,0 ug de REV, e 8,4? G de glicoproteína do vírus da estomatite vesicular (VSVG) para o reagente de transfecção / reduced mistura meio de soro. </li><li> Incubar a mistura durante 20 min à temperatura ambiente. </li><li> Substituir o meio de crescimento com 16 ml de 10% de FBS em DMEM. </li><li> Adicionar a mistura de ADN / reagente de transfecção / meio de soro reduzido, gota a gota para o prato. </li><li> Agite ligeiramente os pratos várias vezes para a frente e para trás seguido por uma direção para a esquerda e para a direita. É normal ver algumas das células HEK293T destacadas neste momento. </li><li> Coloque o prato na incubadora. </li><li> Após 24 horas de incubação, substituir a mídia no prato com 25 ml de meio de crescimento. </li><li> No dia seguinte, recolher o meio de crescimento contendo o lentivírus (primeira coleção). </li><li> Repita o passo 2.2.10 para coletar mais lentivírus (a segunda cobrança). </li><li> Reúnem-se os primeiro e o segundo recolha, e remover os detritos celulares por centrifugação (2500 xg durante 5 min). </li><li> Filtrar a coleção com um filtro descartável garrafa-top (0,45 poro), econcentra-se o vírus por centrifugação a recolha a 31.400 xg durante 2 horas à temperatura ambiente (rotor JA-20). Marcar a parte inferior do tubo de centrifugação em que o pelete é localizado. Decantar o sobrenadante, e deixar o tubo invertido à TA durante cerca de 15 min. </li><li> Ressuspender o sedimento contendo vírus com 100 ul de PBS 1X. Evite bolhas. </li><li> Armazenar o vírus concentrada a -80 ° C. Evite ciclos de congelamento e descongelamento. </li><li> Determinar a quantidade óptima de vírus necessária para transduzir as células Caco-2 por meio de titulação. Para poupar custos, utilizar uma placa de 24 poços (0,3 ml de meio de crescimento / poço) em vez de um 10 centímetros prato de cultura de tecidos para a titulação. <ol><li> Adicionar quantidades crescentes do vírus concentrado (0, 1, 2, 5, 10, 25, e 50 uL / ​​poço) suplementado com polibreno (concentração final = 5 ug / ml) para a 40-70% confluentes células Caco-2. Agitar a placa de 24 poços como descrito em suavemente 1.1.2. </li><li> Monitore sua expressão gênica (Passo 2.1.10.) (Figura 2 fundo4 painéis). </li></ol></li><li> Desde a expressão de transgene é geralmente sustentado, manter as células transduzidas para experiências futuras e monitorar continuamente a sua expressão gênica (Passo 2.1.10.). </li></ol></li></ol> 3. Estimular o Lipoproteína Secreção <ol><li> Usando o prato de cultura de tecido normal <ol><li> Prepara-se uma mistura de lípidos de 100X através da mistura de 50 mg de ácido oleico, 40 mg de lecitina, e 48 mg de taurocolato de sódio. Levar o volume a 900 mL com PBS (o suficiente para pratos 8). Vortex vigorosamente a mistura de lípidos. </li><li> Adicionar 900 ul da mistura de lípidos 100X em 89,1 ml de meio de crescimento. Misturar e filtrar a mídia contendo lípidos. As concentrações finais (1x) de ácido oleico, lecitina, e taurocolato de sódio é 2,0 mM, 1,36 mM e 1,0 mM, respectivamente. </li><li> Adicionar 10 ml de meio contendo lípido para as células. </li><li> Incubam-se as células com meio contendo o lípido durante 4 horas num 37° C incubadora. </li><li> Lavam-se as células com PBS três vezes. </li><li> Adicionam-se 10 ml do meio de crescimento para as células para recolher a secreção de lipoproteínas. </li><li> Incubar durante 2 horas num banho a 37 ° C incubadora. </li><li> Recolhe a mídia contendo lipoproteína. </li></ol></li><li> Usando o sistema de membrana permeável <ol><li> Siga os passos 3.1.1-3.1.2 para preparar a mistura de lípidos. </li><li> Adicionar 10 ml de meio contendo lípido para a câmara de apical e 10 ml de meio de crescimento para a câmara basolateral. </li><li> Incuba-se durante 4 horas numa incubadora de 37 ° C. Recolhe a mídia contendo lipoproteína na câmara basolateral. </li></ol></li></ol> 4. Isolamento de lipoproteína (Figura 3) <ol><li> Remover os restos celulares do meio contendo lipoproteína por centrifugação (2000 xg durante 5 min). </li><li> Decantar a mídia em um tubo de 50 ml. </li><li> Adicionar 5,95 g de cloreto de sódio para a mídia. </li><li> Se a sample serão analisados ​​pelo MET, adicionar um inibidor da protease de cocktail comprimido para manter a integridade das lipoproteínas. </li><li> Levar o volume a 23 ml com água (1,2 g / ml de solução de NaCl a densidade). </li><li> Dissolve-se os solutos completamente. </li><li> Decantar a totalidade da solução para um tubo de ultracentrífuga policarbonato. </li><li> Suavemente sobrepor a solução densidade 1,2 g / ml com 0,5 ml de água (1,0 g / ml de densidade). </li><li> Equilibrar o tubo por peso e não por volume. </li><li> Carregue suavemente os tubos no rotor T865. </li><li> Rodar as amostras na ultracentrífuga a 429.460 xg durante 24 horas a 4 ° C. </li><li> Isolar imediatamente o topo 0,5 ml de solução por pipetagem suave. Mantenha o tubo o mais imóvel possível. </li></ol> 5. Análise TEM <ol><li> Prepare 5 ml de ácido fosfotúngstico a 2% (w / v) e ajustar o pH a 6,0. </li><li> Filtra-se o ácido fosfotúngstico a 2% com um filtro de seringa (tamanho de poro: 0,2 um). </li><li> Gota de 20 ul da amostra em um prato de lipoproteína estéril. </li><li> Gota de 20 ul de ácido fosfotúngstico a 2% filtrada ao lado da amostra no mesmo prato. </li><li> Suavemente soltar uma grade EM com o lado maçante descansando na amostra. </li><li> Incubar à temperatura ambiente durante 1 min. </li><li> Bater levemente o lado da grade de EM num papel de filtro para remover a amostra da rede. </li><li> Suavemente soltar a grade EM com o lado maçante descansando no ácido fosfotúngstico 2%. </li><li> Incubar à temperatura ambiente durante 1 min. </li><li> Bater levemente o lado da grade de EM num papel de filtro para remover o ácido fosfotúngstico a partir da rede. </li><li> Usando uma ETM, capturar as imagens das lipoproteínas (Figura 4). </li></ol>

<ol>
	<li>Drover, V. 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=CD36+deficiency+impairs+intestinal+lipid+secretion+and+clearance+of+chylomicrons+from+the+blood.">CD36 deficiency impairs intestinal lipid secretion and clearance of chylomicrons from the blood.</a> J Clin Invest. 115 (5), 1290-1297 (2005).</li><li>Nauli, A. 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=CD36+is+important+for+chylomicron+formation+and+secretion+and+may+mediate+cholesterol+uptake+in+the+proximal+intestine.">CD36 is important for chylomicron formation and secretion and may mediate cholesterol uptake in the proximal intestine.</a> Gastroenterology. 131 (4), 1197-1207 (2006).</li><li>Nauli, A. M., Zheng, S., Yang, Q., Li, R., Jandacek, R., Tso, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+alkaline+phosphatase+release+is+not+associated+with+chylomicron+formation.">Intestinal alkaline phosphatase release is not associated with chylomicron formation.</a> Am J Physiol Gastrointest Liver Physiol. 284 (4), G583-G587 (2003).</li><li>Lo, C. -. 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=Why+does+the+gut+choose+apolipoprotein+B48+but+not+B100+for+chylomicron+formation.">Why does the gut choose apolipoprotein B48 but not B100 for chylomicron formation.</a> Am J Physiol Gastrointest Liver Physiol. 294 (1), G344-G352 (2008).</li><li>Jandacek, R. J., Heubi, J. E., Tso, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel,+noninvasive+method+for+the+measurement+of+intestinal+fat+absorption.">A novel, noninvasive method for the measurement of intestinal fat absorption.</a> Gastroenterology. 127 (1), 139-144 (2004).</li><li>Nauli, A. M., Nauli, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+transport+as+a+potential+determinant+of+drug+bioavailability.">Intestinal transport as a potential determinant of drug bioavailability.</a> Curr Clin Pharmacol. 8 (3), 247-255 (2013).</li><li>Tso, P., Nauli, A., Lo, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enterocyte+fatty+acid+uptake+and+intestinal+fatty+acid-binding+protein.">Enterocyte fatty acid uptake and intestinal fatty acid-binding protein.</a> Biochem Soc Trans. 32 (Pt 1), 75-78 (2004).</li><li>Nauli, A. M., Sun, Y., Whittimore, J. D., Atyia, S., Krishnaswamy, G., Nauli, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chylomicrons+produced+by+Caco-2+cells+contained+ApoB-48+with+diameter+of+80-200+nm.">Chylomicrons produced by Caco-2 cells contained ApoB-48 with diameter of 80-200 nm.</a> Physiol Rep. 2 (6), (2014).</li><li>Rubinson, D. 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=A+lentivirus-based+system+to+functionally+silence+genes+in+primary+mammalian+cells,+stem+cells+and+transgenic+mice+by+RNA+interference.">A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference.</a> Nat Genet. 33 (3), 401-406 (2003).</li><li>Li, M., Husic, N., Lin, Y., Snider, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+lentiviral+vectors+for+transducing+cells+from+the+central+nervous+system.">Production of lentiviral vectors for transducing cells from the central nervous system.</a> J Vis Exp. (63), e4031 (2012).</li><li>Pottekat, 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=Insulin+biosynthetic+interaction+network+component,+TMEM24,+facilitates+insulin+reserve+pool+release.">Insulin biosynthetic interaction network component, TMEM24, facilitates insulin reserve pool release.</a> Cell Rep. 4 (5), 921-930 (2013).</li><li>Van Greevenbroek, M. M., van Meer, G., Erkelens, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+saturated,+mono-,+and+polyunsaturated+fatty+acids+on+the+secretion+of+apo+B+containing+lipoproteins+by+Caco-2+cells.">Effects of saturated, mono-, and polyunsaturated fatty acids on the secretion of apo B containing lipoproteins by Caco-2 cells.</a> Atherosclerosis. 21 (1), 139-150 (1996).</li><li>Levy, E., Yotov, W., Seidman, E. G., Garofalo, C., Delvin, E., M&#233;nard, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caco-2+cells+and+human+fetal+colon:+a+comparative+analysis+of+their+lipid+transport.">Caco-2 cells and human fetal colon: a comparative analysis of their lipid transport.</a> Biochim Biophys Acta. 1439 (3), 353-362 (1999).</li><li>Luchoomun, J., Hussain, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+and+secretion+of+chylomicrons+by+differentiated+Caco-2+cells.+Nascent+triglycerides+and+preformed+phospholipids+are+preferentially+used+for+lipoprotein+assembly.">Assembly and secretion of chylomicrons by differentiated Caco-2 cells. Nascent triglycerides and preformed phospholipids are preferentially used for lipoprotein assembly.</a> J Biol Chem. 274 (28), 19565-19572 (1999).</li></ol>

The author has nothing to disclose.

Neste artigo, os dois sistemas que podem ser utilizados para manter células Caco-2 são descritos, a saber, o prato de cultura de tecidos normal e a membrana permeável. Os benefícios de usar o sistema de membrana permeável incluem a separação do apical e basolateral os compartimentos, e a capacidade de incubar a mistura de lípidos e recolher a secreção de lipoproteínas simultaneamente. No entanto, as pastilhas de membranas permeáveis ​​são caros, e a sua membrana de policarbonato não permite uma boa vis...

Estudos de absorção intestinal de gordura da dieta, e drogas e vitaminas solúveis em lípidos pode ser realizada in vivo usando um modelo de fistula linfática 1-4. No entanto, as técnicas cirúrgicas envolvidas não são apenas um desafio, mas também caro. Embora abordagens in vivo com base na análise fecal podem ser utilizados, eles são utilizados principalmente para determinar a percentagem de absorção pelo tracto gastrointestinal 2,5. O modelo in vitro descrito neste trabalho é mais rentável, e as técnicas envolvidas são, indiscutivelmente, menos desafiador. Estudos de modificação genética são também mais económico e menos quando eles são conduzidos usando este modelo in vitro demorado. Uma vez que os materiais solúveis em lípidos que são absorvidos por enterócitos são empacotados em lipoproteínas de 6,7, a eficácia deste modelo in vitro para a produção de lipoproteínas é crucial. Os dois intestina principall lipoproteínas são quilomicrons e lipoproteínas de muito baixa densidade (VLDL). Os quilomicrons, lipoproteínas como definidas com 80 nm ou mais de diâmetro, são produzidos de forma rigorosa o intestino delgado, quando lípidos estão abundantemente presentes no lúmen gastrointestinal. Uma vez que eles são os maiores lipoproteínas, chylomicrons são concebivelmente os transportadores lipídicos mais eficientes. Este modelo in vitro, que é capaz de produzir quilomicrons 8, pode ser usado para estudar a absorção de gordura dietética, a vitamina solúvel em lípidos absorção pelo intestino, e biodisponibilidade oral da droga lipofílica. A presença de moléculas solúveis em lípidos, vitaminas ou drogas na fracção de lipoproteínas é um indicador da sua absorção pelo intestino delgado. Como discutido anteriormente, este modelo pode ser usado para melhorar a biodisponibilidade oral da droga lipofílica 6. Este artigo descreve como células Caco-2 devem ser mantidas na membrana permeável ou regular pratos de cultura de tecidos, como os lipídios mixture para estimular a produção de lipoproteína deve ser preparado, como o sistema de expressão de lentivírus pode ser empregue para atingir a sobre-expressão eficaz, e como as lipoproteínas isolados devem ser analisados.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>DMEM</td>
			<td>VWR</td>
			<td>16750-112</td>
			<td>Pre-warm the growth media in individual tissue culture dish before adding cells</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Fisher</td>
			<td>3600511</td>
			<td>We do not heat inactivate our serum</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>VWR</td>
			<td>45000-660</td>
			<td>Cells can be washed with PBS prior to trypsin treatment&#160;</td>
		</tr>
		<tr>
			<td>Permeable membrane system</td>
			<td>Fisher</td>
			<td>7200173</td>
			<td>10-cm dish, 3-mm pore size, polycarbonate membrane</td>
		</tr>
		<tr>
			<td>10 cm dish</td>
			<td>Fisher</td>
			<td>08-772-E</td>
			<td>Tissue culture dish</td>
		</tr>
		<tr>
			<td>15 cm dish</td>
			<td>Fisher</td>
			<td>08-772-24</td>
			<td>Tissue culture dish</td>
		</tr>
		<tr>
			<td>24 well plate</td>
			<td>Fisher</td>
			<td>12565163</td>
			<td>Tissue culture plate</td>
		</tr>
		<tr>
			<td>Lipid-based transfection reagent</td>
			<td>Fisher</td>
			<td>PRE2691</td>
			<td>Can be substituted with other transfection reagent</td>
		</tr>
		<tr>
			<td>Reduced serum media</td>
			<td>Invitrogen</td>
			<td>11058021</td>
			<td>For transfection</td>
		</tr>
		<tr>
			<td>pLL3.7 eGFP</td>
			<td>Addgene</td>
			<td>11795</td>
			<td><a href="https://www.addgene.org/11795/">https://www.addgene.org/11795/</a></td>
		</tr>
		<tr>
			<td>Bottle-top filter</td>
			<td>Fisher</td>
			<td>9761120</td>
			<td>0.45 mm pore</td>
		</tr>
		<tr>
			<td>Polybrene</td>
			<td>Fisher</td>
			<td>NC9840454</td>
			<td>10 mg/mL</td>
		</tr>
		<tr>
			<td>Oleic acid</td>
			<td>Sigma</td>
			<td>01383-5G</td>
			<td>Prevent freeze-thaw cycle</td>
		</tr>
		<tr>
			<td>Lecithin&#160;</td>
			<td>Fisher</td>
			<td>IC10214625</td>
			<td>Egg lecithin&#160;</td>
		</tr>
		<tr>
			<td>Sodium taurocholate</td>
			<td>Fisher</td>
			<td>NC9620276</td>
			<td>Product discontinued; alternative catalog number: 50-121-7956</td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail tablet (EDTA-free)</td>
			<td>Fisher</td>
			<td>5892791001</td>
			<td>Used mainly for samples that need TEM analysis</td>
		</tr>
		<tr>
			<td>Polycarbonate ultracentrifuge tube</td>
			<td>Fisher</td>
			<td>NC9696153</td>
			<td>Reusing it multiple times will collapse the tube</td>
		</tr>
		<tr>
			<td>Lid for ultracentrifuge tube</td>
			<td>Fisher</td>
			<td>NC9796914</td>
			<td>A tool is required to remove the tube/lid from rotor</td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>Fisher</td>
			<td>50-949-261</td>
			<td>Disposable</td>
		</tr>
		<tr>
			<td>Syringe filter</td>
			<td>Fisher</td>
			<td>09-719C</td>
			<td>Pore size = 0.2 mm; nylon</td>
		</tr>
		<tr>
			<td>Phosphotungstic acid</td>
			<td>Fisher</td>
			<td>AC208310250</td>
			<td>For preparing 2% phosphotungstic acid, pH 6.0</td>
		</tr>
		<tr>
			<td>Tweezer</td>
			<td>Fisher</td>
			<td>50-238-62</td>
			<td>Extra fine and strong tips</td>
		</tr>
		<tr>
			<td>Formvar/carbon grid</td>
			<td>Fisher</td>
			<td>50-260-34</td>
			<td>Formvar/carbon film square grid 400 Copper</td>
		</tr>
	</tbody>
</table>

using caco-2 cells to study lipid transport by the intestine

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely accepted as the in vivo model of dietary fat absorption, the surgical techniques involved are challenging and expensive. Genetic manipulation of the animal model is also costly and time consuming. The alternative in vitro model is arguably more affordable, timesaving, and less challenging. Importantly, the in vitro model allows investigators to examine the enterocytes as an isolated system, reducing the complexity inherent in the whole organism model. This paper describes how human colon carcinoma cells (Caco-2) can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs and vitamins. It explains the proper maintenance of Caco-2 cells and the preparation of their lipid mixture; and it further discusses the valuable option of using the permeable membrane system. Since differentiated Caco-2 cells are polarized, the main advantage of using the permeable membrane system is that it separates the apical from the basolateral compartment. Consequently, the lipid mixture can be added to the apical compartment while the lipoproteins can be collected from the basolateral compartment. In addition, the effectiveness of the lentivirus expression system in upregulating gene expression in Caco-2 cells is discussed. Lastly, this paper describes how to confirm the successful isolation of intestinal lipoproteins by transmission electron microscopy (TEM).

Figura 1 apresenta normais de 13 dias pós-confluentes células Caco-2. O aparecimento de estruturas em forma de cúpula e gotículas lipídicas intracelulares são características diferenciadas de células Caco-2. Quando as células Caco-2 não são dispersos igualmente durante a sementeira, eles vão aglutinar-se e crescer demais em certas áreas do prato; e haverá algumas áreas no prato sem quaisquer células. Roda e colocando o prato numa superfície inclinada deve ser evitada. Também é importa...

Watch this Scientific Journal Video about Using Caco-2 Cells to Study Lipid Transport by the Intestine at JoVE.com

Using Caco-2 Cells to Study Lipid Transport by the Intestine

Caco-2 cells can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs/vitamins. The permeable membrane system separates the apical from the basolateral compartment, while the lentivirus expression system offers an effective gene overexpression method. The isolation of lipoproteins is confirmed by TEM.

Usando Caco-2 em células para estudo LIPID Transportes pelo intestino

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely ...

using-caco-2-cells-to-study-lipid-transport-by-the-intestine

Research

JoVE Journal

Biology

18.7K visualizações.  California Northstate University. Caco-2 cells can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs/vitamins. The permeable membrane system separates the apical from the basolateral compartment, while the lentivirus expression system offers an effective gene overexpression method. The isolation of lipoproteins is confirmed by TEM.

Vídeo: Using Caco-2 Cells to Study Lipid Transport by the Intestine

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Teste de sensibilidade visual para a velocidade e direção do movimento em Lagartos

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Biologia

Computer-Generated Animal Model Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In particular, understanding the constraints on visual signal design for communication has been of great interest. Traditional methods for investigating animal interactions have used basic observational techniques, staged encounters, or physical manipulation of morphology. Less intrusive methods have tried to simulate conspecifics using crude playback tools, such as mirrors, still images, or models. As technology has become more advanced, video playback has emerged as another tool in which to examine visual communication (Rosenthal, 2000). However, to move one step further, the application of computer-animation now allows researchers to specifically isolate critical components necessary to elicit social responses from conspecifics, and manipulate these features to control interactions. Here, I provide detail on how to create an animation using the Jacky dragon as a model, but this process may be adaptable for other species. In building the animation, I elected to use Lightwave  3D to alter object morphology, add texture, install bones, and provide comparable weight shading that prevents exaggerated movement. The animation is then matched to select motor patterns to replicate critical movement features. Finally, the sequence must rendered into an individual clip for presentation. Although there are other adaptable techniques, this particular method had been demonstrated to be effective in eliciting both conspicuous and social responses in staged interactions.

Computer-generated stimuli using the Jacky dragon as a model.

Gerados por computador Modelo Estímulos animal

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite morphogenesis at the functional and molecular levels. To aid in these analyses, we have developed a strategy for the isolation of a subclass of PNS neurons called dendritic arborization (da) neurons that have been widely used for studying dendrite morphogenesis1,2. These neurons are very difficult to isolate as a pure population, due in part to their extremely low occurrence and their difficult-to-reach location below the tough chitinous larval cuticle. Our newly developed method overcomes these challenges, and is based on a fast and specific cell enrichment using antibody-coated magnetic beads. For our magnetic bead sorting studies, we have used age-matched third instar larvae expressing a mouse CD8 tagged GFP fusion protein (UAS-mCD8-GFP)3 under the control of either the class IV dendritic arborization (da) neuron-specific pickpocket (ppk)-GAL4 driver4 or the control of the pan-da neuron-specific GAL421-7 driver5. Although this protocol has been optimized for isolating PNS cells which are attached to the inner wall of the larval cuticle, by varying a few parameters, the same protocol could be used to isolate many different cell types attached to the cuticle at larval or pupal stages of development (e.g. epithelia, muscle, oenocytes etc.), or other cell types from larval organs depending upon the GAL4-specific driver expression pattern. The RNA isolated by this method is of high quality and can be readily used for downstream genomic analyses such as microarray gene expression profiling studies. This approach offers a powerful new tool to perform studies on isolated Drosophila dendritic arborization (da) neurons thereby providing novel insights into the molecular mechanisms underlying dendrite morphogenesis.

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

In this video-article we present a method for the isolation and purification of Drosophila peripheral neurons using a fast magnetic bead assisted cell sorting strategy. RNA obtained from the isolated cells can be readily used for downstream applications including microarray analyses.

Isolamento e purificação de Drosophila Neurônios periféricos por classificação Bead Magnética

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite ...

Using the optokinetic response to study visual function of zebrafish

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the visual functions of larval zebrafish1-5. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adult zabrafish. Here, we introduce how to measure the OKR of adult zebrafish with our simple custom-built apparatus using a new protocol which is established in our lab. Both our apparatus and step-by-step procedure of OKR in adult zebrafish are illustrated in this video. In addition, the measurements of the larval OKR, as well as the optomotor response (OMR) test of adult zebrafish, are also demonstrated in this video. This OKR assay of adult zebrafish in our experiment may last for up to 4 hours. Such OKR test applied in adult fish will benefit to visual function investigation more efficiently when the adult fish vision system is manipulated.
Su-Qi Zou and Wu Yin contributed equally to this paper.

Optokinetic response has been widely used to assess the visual functions of larval zebrafish. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adults1-5. Here, we introduce how to measure the OKR of adult zebrafish using a new protocol which is established in our lab.

Usando a resposta optocinético para estudar a função visual de zebrafish

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the ...

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

Whereas cation transport by the electrogenic membrane transporter Na+,K+-ATPase can be measured by electrophysiology, the electroneutrally operating gastric H+,K+-ATPase is more difficult to investigate. Many transport assays utilize radioisotopes to achieve a sufficient signal-to-noise ratio, however, the necessary security measures impose severe restrictions regarding human exposure or assay design. Furthermore, ion transport across cell membranes is critically influenced by the membrane potential, which is not straightforwardly controlled in cell culture or in proteoliposome preparations. Here, we make use of the outstanding sensitivity of atomic absorption spectrophotometry (AAS) towards trace amounts of chemical elements to measure Rb+ or Li+ transport by Na+,K+- or gastric H+,K+-ATPase in single cells. Using Xenopus oocytes as expression system, we determine the amount of Rb+ (Li+) transported into the cells by measuring samples of single-oocyte homogenates in an AAS device equipped with a transversely heated graphite atomizer (THGA) furnace, which is loaded from an autosampler. Since the background of unspecific Rb+ uptake into control oocytes or during application of ATPase-specific inhibitors is very small, it is possible to implement complex kinetic assay schemes involving a large number of experimental conditions simultaneously, or to compare the transport capacity and kinetics of site-specifically mutated transporters with high precision. Furthermore, since cation uptake is determined on single cells, the flux experiments can be carried out in combination with two-electrode voltage-clamping (TEVC) to achieve accurate control of the membrane potential and current. This allowed e.g. to quantitatively determine the 3Na+/2K+ transport stoichiometry of the Na+,K+-ATPase and enabled for the first time to investigate the voltage dependence of cation transport by the electroneutrally operating gastric H+,K+-ATPase. In principle, the assay is not limited to K+-transporting membrane proteins, but it may work equally well to address the activity of heavy or transition metal transporters, or uptake of chemical elements by endocytotic processes.

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

We describe a method to quantify the activity of K+-countertransporting P-type ATPases by heterologous expression of the enzymes in Xenopus oocytes and measuring Rb+ or Li+ uptake into individual cells by atomic absorption spectrophotometry. The method is a sensitive and safe alternative to radioisotope flux experiments facilitating complex kinetic studies.

Medindo o transporte de catiões de Na, K e H, K-ATPase em<em&gt; Xenopus</em&gt; Oócitos por espectrofotometria de absorção atômica: uma alternativa para Ensaios de Radioisótopos

Whereas cation transport by the electrogenic membrane transporter Na+,K+-ATPase can be measured by electrophysiology, the electroneutrally operating ...

Methods to Study Epithelial Transport Protein Function and Expression in Native Intestine and Caco-2 Cells Grown in 3D

The intestinal epithelium has important transport and barrier functions that play key roles in normal physiological functions of the body while providing a barrier to foreign particles. Impaired epithelial transport (ion, nutrient, or drugs) has been associated with many diseases and can have consequences that extend beyond the normal physiological functions of the transporters, such as by influencing epithelial integrity and the gut microbiome. Understanding the function and regulation of transport proteins is critical for the development of improved therapeutic interventions. The biggest challenge in the study of epithelial transport is developing a suitable model system that recapitulates important features of the native intestinal epithelial cells. Several in vitro cell culture models, such as Caco-2, T-84, and HT-29-Cl.19A cells are typically used in epithelial transport research. These cell lines represent a reductionist approach to modeling the epithelium and have been used in many mechanistic studies, including their examination of epithelial-microbial interactions. However, cell monolayers do not accurately reflect cell-cell interactions and the in vivo microenvironment. Cells grown in 3D have shown to be promising models for drug permeability studies. We show that Caco-2 cells in 3D can be used to study epithelial transporters. It is also important that studies in Caco-2 cells are complemented with other models to rule out cell specific effects and to take into account the complexity of the native intestine. Several methods have been previously used to assess the functionality of transporters, such as everted sac and uptake in isolated epithelial cells or in isolated plasma membrane vesicles. Taking into consideration the challenges in the field with respect to models and the measurement of transport function, we demonstrate here a protocol to grow Caco-2 cells in 3D and describe the use of an Ussing chamber as an effective approach to measure serotonin transport, such as in intact polarized intestinal epithelia.

We describe simple methods to study the regulation of intestinal serotonin transporter (SERT) function and expression using an in vitro cell culture model of Caco-2 cells grown in 3D&#160;and an ex vivo model of mouse intestines. These methods are applicable to the study of other epithelial transporters.

Métodos de estudo epitelial Transporte função das proteínas e Expressão em Native Intestino e Caco-2 células cultivadas em 3D

The intestinal epithelium has important transport and barrier functions that play key roles in normal physiological functions of the body while providing ...

Mixed Primary Cultures of Murine Small Intestine Intended for the Study of Gut Hormone Secretion and Live Cell Imaging of Enteroendocrine Cells

The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal epithelium. Despite their physiological importance, enteroendocrine cells represent only a small fraction of the epithelial cell population and in the past, their characterization has presented a considerable challenge resulting in a reliance on cell line models. Here, we provide a detailed protocol for the isolation and culture of mixed murine small intestinal cells. These primary cultures have been used to identify the signaling pathways underlying the stimulation and inhibition of gut peptide secretion in response to a number of nutrients and neuropeptides as well as pharmacological agents. Furthermore, in combination with the use of transgenic fluorescent reporter mice, we have demonstrated that these primary cultures become a powerful tool for the examination of fluorescently-tagged enteroendocrine cells at the intracellular level, using methods such as patch clamping and single-cell calcium and cAMP-FRET imaging.

This protocol describes the isolation and culture of mixed murine small intestinal cells. These primary intestinal cultures enable the investigation of signaling pathways underlying gut peptide secretion using a number of techniques.

Culturas primárias mistas de Murino Intestino Delgado destinadas ao estudo de Gut hormonal Secreção e imagens de células vivas de Células Enteroendócrinas

The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal ...

Tethered Bilayer Lipid Membranes to Monitor Heat Transfer between Gold Nanoparticles and Lipid Membranes

Here we report a protocol to investigate the heat transfer between irradiated gold nanoparticles (GNPs) and bilayer lipid membranes by electrochemistry using tethered bilayer lipid membranes (tBLMs) assembled on gold electrodes. Irradiated modified GNPs, such as streptavidin-conjugated GNPs, are embedded in tBLMs containing target molecules, such as biotin. By using this approach, the heat transfer processes between irradiated GNPs and model bilayer lipid membrane with entities of interest are mediated by a horizontally focused laser beam. The thermal predictive computational model is used to confirm the electrochemically induced conductance changes in the tBLMs. Under the specific conditions used, detecting heat pulses required specific attachment of the gold nanoparticles to the membrane surface, while unbound gold nanoparticles failed to elicit a measurable response. This technique serves as a powerful detection biosensor which can be directly utilized for the design and development of strategies for thermal therapies that permits optimization of the laser parameters, particle size, particle coatings and composition.

This work outlines a protocol to achieve dynamic, non-invasive monitoring of heat transfer from laser-irradiated gold nanoparticles to tBLMs. The system combines impedance spectroscopy for the real-time measurement of conductance changes across the tBLMs, with a horizontally focused laser beam that drives gold nanoparticle illumination, for heat production.

Membranas lipídicas de bicamadas tethered para monitorar a transferência de calor entre nanopartículas de ouro e membranas lipídicas

Here we report a protocol to investigate the heat transfer between irradiated gold nanoparticles (GNPs) and bilayer lipid membranes by electrochemistry ...

The Caco-2 Cell Bioassay for Measurement of Food Iron Bioavailability

Knowledge of Fe bioavailability is critical to the assessment of the nutritional quality of Fe in foods. In vivo measurement of Fe bioavailability is limited by cost, throughput, and the caveats inherent to isotopic labeling of the food Fe. Thus, there exists a critical need for an approach that is high-throughput and cost-effective. The Caco-2 cell bioassay was developed to satisfy this need. The Caco-2 cell bioassay for Fe bioavailability utilizes simulated gastric and intestinal digestion coupled with culture of a human intestinal epithelial cell line known as Caco-2. In Caco-2 cells, Fe uptake stimulates the intracellular formation of ferritin, an Fe storage protein easily measured by enzyme-linked immunosorbent assay (ELISA). Ferritin forms in proportion to Fe uptake; thus, by measuring Caco-2 cell ferritin production, one can assess intestinal Fe uptake from simulated food digests into the enterocyte. 
Via this approach, the model replicates the key initial step that determines food Fe bioavailability. Since its inception in 1998, this model approach has been rigorously compared to factors known to influence human Fe bioavailability. Moreover, it has been applied in parallel studies, with three human efficacy studies evaluating Fe biofortified crops. In all cases, the bioassay correctly predicted the relative amounts of Fe bioavailability from the factors, crops, and overall diet. This paper provides detailed methods on Caco-2 cell culture coupled with the in vitro digestion process and cell ferritin ELISA necessary to conduct the Caco-2 cell bioassay for Fe bioavailability.

The Caco-2 cell bioassay for iron (Fe) bioavailability represents a cost-effective and versatile approach to assess Fe bioavailability from foods, food products, supplements, meals, and even diet regimens. Thoroughly validated to human studies, it represents the state of the art for studies of Fe bioavailability.

O Bioensaio celular Caco-2 para Medição da Biodisponibilidade de Ferro Alimentar

Knowledge of Fe bioavailability is critical to the assessment of the nutritional quality of Fe in foods. In vivo measurement of Fe bioavailability is ...

Sample Preparation for Rapid Lipid Analysis in Drosophila Brain Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging

Lipid profiling, or lipidomics, is a well-established technique used to study the entire lipid content of a cell or tissue. Information acquired from&#160;lipidomics is valuable in studying the pathways involved in development, disease, and cellular metabolism. Many tools and instrumentations have aided lipidomics projects, most notably various combinations of mass spectrometry and liquid chromatography techniques. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) has recently emerged as a powerful imaging technique that complements conventional approaches. This novel technique provides unique information on the spatial distribution of lipids within tissue compartments, which was previously unattainable without the use of excessive modifications. The sample preparation of the MALDI MSI approach is critical and, therefore, is the focus of this paper. This paper presents a rapid lipid analysis of a large number of Drosophila brains embedded in optimal cutting temperature compound (OCT) to provide a detailed protocol for the preparation of small tissues for lipid analysis or metabolite and small molecule analysis through MALDI MSI.

Sample Preparation for Rapid Lipid Analysis in Drosophila Brain Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging

The aim of this protocol is to provide detailed guidance on the proper sample preparation for lipid and metabolite analysis in small tissues, such as the Drosophila brain, using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging.

Preparação amostral para análise rápida lipídica no cérebro de Drosophila usando desorção a laser assistida por matriz/imagem de espectrometria de massa

Usando Caco-2 em células para estudo LIPID Transportes pelo intestino

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