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. Mantenimiento de las células Caco-2 <ol><li> El uso de platos regulares de cultivo de tejidos <ol><li> Descongelar las 2-Caco células de un vial congelado colocando el vial en un baño de agua a 37 ° C, e inmediatamente añadirlos a una placa de cultivo tisular de 10 cm que contenía 10 ml de medio de crecimiento (suero bovino fetal pre-calentado 15% (FBS ) en Medio Eagle Modificado de Dulbecco (DMEM)). <ol><li> Cuando las células Caco-2 han llegado a 50-70% de confluencia, ellos dividido 1: 6 mediante la incubación de las células con 3 ml de 0,05% / 0,53 mM de ácido etilendiaminotetraacético Tripsina (EDTA) a 37 ° C hasta que se separan (15 min) . Para evitar la formación de grumos de células, mezclar las células varias veces por pipeteo suave. Añadir 0,5 ml de las células tripsinizadas a una placa de cultivo de tejido de 10 cm que contenía 10 ml de medio de crecimiento pre-calentado. </li></ol></li><li> Agitar suavemente los platos varias veces en una dirección hacia adelante y hacia atrás seguido de una dirección hacia la izquierda y hacia la derecha. Evite remolinos de los platosya que puede resultar en una dispersión de células desigual. </li><li> Colocar las cápsulas en una superficie plana en una incubadora a 37 ° se suministra con 5% de CO 2. Superficies inclinadas también pueden resultar en una dispersión de células desigual. </li><li> Cambie los medios de crecimiento de un día después de comenzar la incubación. </li><li> Monitorear las células y registrar el día que lleguen a la confluencia. Tomará alrededor de 1 semana para que las células alcanzan la confluencia del día en que se dividen. </li><li> Cambiar el medio de crecimiento dos veces a la semana antes de que las células alcanzan la confluencia. Una vez que las células han alcanzado la confluencia, cambiar los medios de cultivo cada dos días. Después de que las células son 7 días post-confluentes, cambiar el medio de crecimiento diario. Nota: Las células están listas para experimentos cuando son 13 días post-confluentes (Figura 1). Dado que las células con el tiempo se vuelven menos efectivos para producir lipoproteínas, utilizar células que se encuentran entre 13 y 17 días post-confluentes 8. Vaya al paso 3 en la forma de realizar el experimento.</li></ol></li><li> Usando el sistema de membrana permeable <ol><li> Semilla las células Caco-2 en el inserto membrana permeable como se describe en el paso 1.1.1.1. arriba. Evitar el uso de células de un vial congelado debido a que su crecimiento celular es normalmente más lenta (período de recuperación). Brevemente, añadir 0,5 ml de las células tratadas con tripsina a la cámara apical (compartimiento superior) que contiene 10 ml de medio de crecimiento pre-calentado. Además, añadir 10 ml de medio de crecimiento pre-calentado a la cámara basolateral (compartimiento inferior). Nota: Para obtener las mejores prácticas, agregue los medios de cultivo a la cámara apical primero y luego a la cámara basolateral. Al retirar los viejos medios, retire los medios basolateral ante los medios apicales. Evite meter la membrana de policarbonato, ya que puede romper la membrana. </li><li> Debido a la visibilidad celular pobres a través de la membrana de policarbonato, sembrar las células Caco-2 en una placa de cultivo de tejidos regular con densidad similar, y el uso de este plato para juzgar la confluencia celular. </li><li> Siga steps 1.1.2-1.1.6 anteriores. </li></ol></li></ol> 2. La sobreexpresión de genes <ol><li> Utilizando el método ordinario transfección <ol><li> Semilla las células Caco-2 en una placa de cultivo de tejidos como se describe en 1.1.1.1.-1.1.4. Nota: Cuando las células son alrededor de 40-50% de confluencia, que están listos para ser transfectadas. </li><li> Añadir 67 l de reactivo de transfección a 472 l de los medios de comunicación séricos reducidos en un tubo de microcentrífuga. Evite el contacto del reactivo de transfección sin diluir con la pared del tubo. </li><li> Añadir 23 g de pLL3.7 aumento de proteína verde fluorescente (EGFP) 9 en el reactivo de transfección / reducido mezcla de medio de suero. </li><li> Incubar la mezcla durante 20 min a TA. </li><li> Reemplace el medio de crecimiento de las células Caco-2 &#39;con 8 ml de 10% de FBS en DMEM. </li><li> Añadir la mezcla de ADN / reactivo de transfección / media suero reducido gota a gota al plato. </li><li> Agite con cuidado los platos varias veces en un delantero y backward dirección seguida por una dirección hacia la izquierda y hacia la derecha. </li><li> Coloque el plato en el 37 ° C incubadora. </li><li> Vuelva a colocar los medios de comunicación en el plato con 10 ml de medio de crecimiento un día después de comenzar la incubación. </li><li> Supervisar la expresión génica. <ol><li> Controlar la expresión génica sin 4 &#39;, 6-diamidino-2-fenilindol (DAPI) tinción. Utilizando un microscopio fluorescente, determinar el porcentaje de células que son verde. El porcentaje representa la eficiencia de transfección. </li><li> Supervisar la expresión génica con DAPI. <ol><li> Lavar las células con solución salina tamponada con fosfato (PBS) tres veces. </li><li> Fijar las células mediante la adición de 10 ml de formaldehído al 4% en PBS (10 min de incubación a RT). </li><li> Lavar las células con PBS tres veces. </li><li> Se incuban las células en la oscuridad durante 15 min a TA con 8 ml de PBS que contenía 1: 500 DAPI, 1% de BSA, 0,01% de digitonina. </li><li> Lavar las células con PBS tres veces. </li><li> Ucantar un microscopio de fluorescencia, determinar el número de células que son verde / azul con relación a los que son de color azul (Figura 2 panel superior). El porcentaje calculado representa la eficiencia de transfección. </li></ol></li></ol></li></ol></li><li> Utilizando el sistema de sobreexpresión lentivirus 10 <ol><li> Semilla de riñón embrionario (HEK) células humanas 293T en un 15 cm placa de cultivo de tejidos (10% de FBS en DMEM como su medio de crecimiento). Nota: Cuando las células son aproximadamente 60-70% de confluencia, que están listos para ser transfectadas. </li><li> Añadir 162 l de reactivo de transfección de 1.133 l de los medios de comunicación séricos reducidos en un tubo de microcentrífuga de 11. Evite el contacto del reactivo de transfección sin diluir con la pared del tubo. </li><li> Añadir 24 g de pLL3.7 eGFP (u otro constructo), 15,6 g de elemento de respuesta Rev (RRE), 6,0 g de REV, y 8,4 g de glicoproteína de virus de la estomatitis vesicular (VSVG) en el reactivo de transfección / rrelucida mezcla de medio de suero. </li><li> Incubar la mezcla durante 20 min a TA. </li><li> Reemplace el medio de crecimiento con 16 ml de 10% de FBS en DMEM. </li><li> Añadir la mezcla de ADN / reactivo de transfección / media suero reducido gota a gota al plato. </li><li> Agitar suavemente los platos varias veces en una dirección hacia adelante y hacia atrás seguido de una dirección hacia la izquierda y hacia la derecha. Es normal ver algunas de las células HEK293T unifamiliares en este punto. </li><li> Coloque el plato en la incubadora. </li><li> Después de 24 h de incubación, sustituir los medios de comunicación en el plato con 25 ml de medio de crecimiento. </li><li> Al día siguiente, recoger el medio de crecimiento que contiene el lentivirus (primera colección). </li><li> Repita el paso 2.2.10 para recoger más lentivirus (la segunda colección). </li><li> Agrupar la primera y la segunda colección, y eliminar los restos celulares por centrifugación (2.500 xg durante 5 min). </li><li> Se filtra la colección con un filtro desechable botella-superior (0,45 micras de poro), yconcentrar el virus por centrifugación de la colección a 31.400 xg durante 2 horas a RT (JA-20 rotor). Marque la parte inferior del tubo de centrífuga donde se encuentra el sedimento. Decantar el sobrenadante, y dejar el tubo invertido a temperatura ambiente durante unos 15 minutos. </li><li> Resuspender el sedimento que contiene el virus con 100 l de 1X PBS. Evite las burbujas. </li><li> Guarde el virus concentrado a -80 ° C. Evite los ciclos de congelación-descongelación. </li><li> Determinar la cantidad óptima de virus necesaria para transducir las células Caco-2 por titulación. Para ahorrar costes, utilizar una placa de 24 pocillos (0,3 ml de crecimiento de los medios de comunicación / pocillo) en lugar de una placa de 10 cm de cultivo de tejidos para la titulación. <ol><li> Añadir cantidades crecientes del virus concentrado (0, 1, 2, 5, 10, 25, y 50 l / pocillo) suplementado con polibreno (concentración final = 5 g / ml) para el 40-70% de confluencia células Caco-2. Agite la placa de 24 pocillos con cuidado como se describe en 1.1.2. </li><li> Vigile su expresión génica (Paso 2.1.10.) (Figura 2 inferior4 paneles). </li></ol></li><li> Dado que la expresión de transgén es generalmente sostenido, mantener las células transducidas para futuros experimentos y controlar continuamente su expresión génica (Paso 2.1.10.). </li></ol></li></ol> 3. Estimular la secreción de lipoproteínas <ol><li> Usando el tejido plato normal cultura <ol><li> Preparar una mezcla de lípidos 100X mezclando 50 mg de ácido oleico, 40 mg de lecitina y 48 mg de taurocolato de sodio. Llevar el volumen a 900 l con PBS (suficiente para 8 platos). Agite la mezcla de lípidos vigorosamente. </li><li> Añadir 900 l de la mezcla de lípidos 100X en 89,1 ml de medio de crecimiento. Mezclar y filtrar los medios de comunicación que contiene lípidos. Las concentraciones finales (1X) de ácido oleico, lecitina, y taurocolato de sodio son 2,0 mM, 1,36 mM, y 1,0 mM, respectivamente. </li><li> Añadir 10 ml de los medios de comunicación que contiene lípidos a las células. </li><li> Se incuban las células con los medios de comunicación que contiene lípidos durante 4 horas en un 37° C incubadora. </li><li> Lavar las células con PBS tres veces. </li><li> Añadir 10 ml de medio de crecimiento a las células para recoger la secreción de lipoproteínas. </li><li> Incubar durante 2 horas en una incubadora a 37 °. </li><li> Se recoge el medio que contiene lipoproteínas. </li></ol></li><li> Usando el sistema de membrana permeable <ol><li> Siga los pasos 3.1.1-3.1.2 para preparar la mezcla de lípidos. </li><li> Añadir 10 ml de los medios de comunicación que contiene lípidos a la cámara apical y 10 ml de medio de crecimiento a la cámara basolateral. </li><li> Incubar durante 4 horas en una incubadora a 37 °. Se recoge el medio que contiene lipoproteínas en la cámara basolateral. </li></ol></li></ol> 4. Aislamiento lipoproteína (Figura 3) <ol><li> Eliminar los restos celulares de los medios de comunicación que contiene lipoproteína mediante centrifugación (2.000 xg durante 5 min). </li><li> Decantar los medios de comunicación en un tubo de 50 ml. </li><li> Añadir 5,95 g de cloruro de sodio a los medios. </li><li> Si la sample será analizada por el TEM, añadir 1 tableta de cóctel inhibidor de proteasa para mantener la integridad de las lipoproteínas. </li><li> Llevar el volumen a 23 ml con agua (1,2 g / ml NaCl solución de densidad). </li><li> Disolver los solutos completamente. </li><li> Decantar toda la solución en un tubo de ultracentrífuga de policarbonato. </li><li> Superponer suavemente la solución de densidad de 1,2 g / ml con 0,5 ml de agua (1,0 g / ml de densidad). </li><li> Equilibrar el tubo en peso y no por volumen. </li><li> Cargar suavemente los tubos en el rotor T865. </li><li> Haga girar las muestras en la ultracentrífuga a 429.460 xg durante 24 horas a 4 ° C. </li><li> Inmediatamente aislar la parte superior solución de 0,5 ml por pipeteo suave. Mantenga el tubo lo más quieto posible. </li></ol> Análisis 5. TEM <ol><li> Preparar 5 ml de ácido fosfotúngstico al 2% (w / v) y ajustar el pH a 6,0. </li><li> Filtrar el ácido fosfotúngstico al 2% con un filtro de jeringa (tamaño de poro: 0,2 micras). </li><li> Caída de 20 l de la muestra de la lipoproteína en un plato estéril. </li><li> Caída de 20 l de ácido fosfotúngstico filtrada 2% junto a la muestra en el mismo plato. </li><li> Caer suavemente una rejilla EM con el lado opaco descansando en la muestra. </li><li> Incubar a temperatura ambiente durante 1 min. </li><li> Golpear suavemente el lado de la rejilla EM sobre un papel de filtro para eliminar la muestra de la red. </li><li> Caer suavemente la rejilla EM con el lado opaco que descansa sobre el ácido fosfotúngstico al 2%. </li><li> Incubar a temperatura ambiente durante 1 min. </li><li> Golpear suavemente el lado de la rejilla EM sobre un papel de filtro para eliminar el ácido fosfotúngstico de la red. </li><li> El uso de un TEM, la captura de las imágenes de las lipoproteínas (Figura 4). </li></ol>

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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. 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The author has nothing to disclose.

En este papel, los dos sistemas que se pueden utilizar para mantener las células Caco-2 se describen, a saber, el plato de cultivo de tejido normal y la membrana permeable. Los beneficios de usar el sistema de membrana permeable incluyen la separación de la apical y basolateral de los compartimentos, y la capacidad de incubar la mezcla de lípidos y recoger la secreción de lipoproteínas de forma simultánea. Sin embargo, los insertos de membrana permeable son caros, y su membrana de policarbonato no permite una buen...

Los estudios de absorción intestinal de grasas en la dieta, y las drogas y vitaminas solubles en lípidos pueden llevar a cabo in vivo mediante el uso de un modelo de fístula linfática de 1 - 4. Sin embargo, las técnicas quirúrgicas que participan no sólo son un reto, pero también costoso. Aunque in vivo enfoques basados ​​en el análisis fecal pueden ser utilizados, que se utilizan principalmente para determinar el porcentaje de absorción por el tracto gastrointestinal 2,5. El modelo in vitro descrito en este artículo es más rentable, y las técnicas utilizadas son sin duda menos difícil. Estudios de modificación genética también son más económicos y menos tiempo cuando se llevaron a cabo utilizando este modelo in vitro. Dado que los materiales solubles en lípidos que son absorbidos por los enterocitos son empaquetados en lipoproteínas de 6,7, la eficacia de este modelo in vitro para producir las lipoproteínas es crucial. Los dos intestina principall lipoproteínas son quilomicrones y lipoproteínas de muy baja densidad (VLDL). Los quilomicrones, definidos como lipoproteínas con 80 nm de diámetro o más, se producen estrictamente por el intestino delgado cuando los lípidos son abundantemente presentes en el lumen gastrointestinal. Puesto que son las lipoproteínas más grandes, los quilomicrones son posiblemente los transportadores de lípidos más eficientes. Este modelo in vitro, que es capaz de producir los quilomicrones 8, se puede utilizar para estudiar la absorción de grasa dietética, soluble en lípidos vitamina absorción por el intestino, y la biodisponibilidad oral de fármaco lipofílico. La presencia de moléculas solubles en lípidos, vitaminas, medicamentos o en la fracción de lipoproteína es un indicador de su absorción por el intestino delgado. Como se discutió anteriormente, este modelo se puede utilizar para mejorar la biodisponibilidad oral de fármaco lipofílico 6. Este artículo describe cómo se deben mantener células Caco-2 en la membrana permeable o platos regulares de cultivo de tejidos, cómo las millas de lípidosxture para estimular la producción de lipoproteínas debe prepararse, cómo el sistema de expresión lentivirus puede ser empleado para lograr la sobreexpresión efectiva, y cómo se debe analizar las lipoproteínas aisladas.

<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 13-días post-confluentes células normales Caco-2 1 muestra. La aparición de estructuras en forma de cúpula y las gotas de lípidos intracelulares son característicos de diferenciadas Caco-2 células. Cuando las células Caco-2 no se dispersan igualmente durante la siembra, se aglutinan y crecer demasiado en ciertas áreas del plato; y habrá algunas áreas en el plato sin células. Remolina y colocando el plato en una superficie inclinada debe ser evitado. También es import...

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.

El uso de células Caco-2 para el Estudio de lípidos Transporte por el 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 vistas.  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.

Video: 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.

Prueba de sensibilidad visual a la velocidad y dirección del movimiento en lagartos

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

Investigación

Biología

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.

Los estímulos generados por ordenador Modelo 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.

Aislamiento y purificación de Drosophila Clasificación de las neuronas periféricas por bolas magnéticas

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 la respuesta de optocinético para estudiar la función visual del pez cebra

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.

Medición de transporte de cationes por Na, K y H, K-ATPasa en<em&gt; Xenopus</em&gt; Los ovocitos mediante espectrofotometría de absorción atómica: una alternativa a ensayos 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 estudio de la función de proteínas de transporte epitelial y expresión en nativo Intestino y Caco-2 células cultivadas en 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.

Mezcla de cultivos primarios de murino intestino delgado previstos para el Estudio de Gut Hormone secreción y Imágenes de células vivas de células enteroendocrinas

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 bicapa atadas para monitorear la transferencia de calor entre las nanopartículas de oro y las 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.

El bioensayo de células Caco-2 para la medición de la biodisponibilidad de hierro en los alimentos

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.

Preparación de muestras para el análisis rápido de lípidos en el cerebro de Drosophila utilizando imágenes de espectrometría de masas de desorción / ionización láser asistidas por matriz

El uso de células Caco-2 para el Estudio de lípidos Transporte por el Intestino

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