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. Manutenzione delle cellule Caco-2 <ol><li> Utilizzando regolari piastre di coltura tissutale <ol><li> Scongelare le cellule Caco-2 da una fiala congelata ponendo la provetta in un bagno d&#39;acqua a 37 °, e subito aggiungerli a un piatto di coltura di tessuto al 10 cm contenente 10 ml di terreni di crescita (siero bovino fetale pre-riscaldato 15% (FBS ) in Dulbecco modificato (DMEM)). <ol><li> Quando le cellule Caco-2 hanno raggiunto 50-70% di confluenza, dividerle 1: 6 incubando le cellule con 3 ml di 0,05% tripsina / 0.53 mM di acido etilendiamminotetraacetico (EDTA) a 37 ° C fino a quando sono staccati (15 min) . Per evitare aggregazione cellulare, mescolare le cellule più volte delicatamente pipettando. Aggiungere 0,5 ml di cellule trypsinized ad un piatto di coltura di tessuto al 10 cm contenente 10 ml di terreni di crescita pre-riscaldato. </li></ol></li><li> Agitare delicatamente i piatti più volte in avanti e indietro, seguito da una direzione verso sinistra e verso destra. Evitare vorticoso i piattiin quanto può provocare una dispersione delle cellule disuguale. </li><li> Porre le capsule su una superficie piana in un incubatore a 37 ° in dotazione con il 5% di CO 2. Superfici inclinate possono anche provocare una dispersione cellulare disuguale. </li><li> Modificare i media di crescita di un giorno dopo l&#39;avvio l&#39;incubazione. </li><li> Monitorare le cellule e registrare la giornata raggiungono confluenza. Ci vorrà circa 1 settimana per le cellule di raggiungere confluenza dal giorno in cui sono divisi. </li><li> Modificare i mezzi di crescita due volte la settimana prima che le cellule raggiungono confluenza. Una volta che le cellule hanno raggiunto confluenza, modificare i mezzi di crescita ogni altro giorno. Dopo che le cellule sono 7 giorni post-confluenti, modificare i mezzi di crescita giornaliera. Nota: Le cellule sono pronte per esperimenti quando sono 13 giorni post-confluenti (Figura 1). Dal momento che le cellule diventeranno meno efficace nel produrre lipoproteine, usare le cellule che si trovano tra i 13 ei 17 giorni dopo confluenti 8. Andare al punto 3 su come condurre l&#39;esperimento.</li></ol></li><li> Utilizzando il sistema di membrana permeabile <ol><li> Seme le cellule Caco-2 nel foglietto membrana permeabile come descritto al punto 1.1.1.1. sopra. Evitare l&#39;uso di cellule da un flaconcino congelato perché la loro crescita cellulare è normalmente più lento (periodo di recupero). Brevemente, aggiungere 0,5 ml delle cellule trypsinized alla camera apicale (scomparto superiore) contenente 10 ml di terreni di crescita pre-riscaldato. Inoltre, aggiungere 10 ml di terreni di crescita pre-riscaldato alla camera basolaterale (scomparto inferiore). Nota: per le migliori prassi, aggiungere i media di crescita alla camera apicale e poi alla camera basolaterale. Durante la rimozione dei vecchi media, rimuovere il supporto basolaterale prima che i media apicali. Evitare colpendo la membrana di policarbonato in quanto potrebbe rompere la membrana. </li><li> Grazie alla visibilità delle cellule poveri attraverso la membrana in policarbonato, seminare le cellule Caco-2 in un piatto di coltura tissutale regolare con densità simile, e utilizzare questo piatto per giudicare la confluenza delle cellule. </li><li> Segui steps 1.1.2-1.1.6 sopra. </li></ol></li></ol> 2. Gene Overexpression <ol><li> Utilizzando l&#39;approccio trasfezione regolare <ol><li> Seme le cellule Caco-2 su un piatto di coltura di tessuti, come descritto nella 1.1.1.1.-1.1.4 sopra. Nota: Quando le cellule sono circa il 40-50% confluenti, sono pronti per essere transfettate. </li><li> Aggiungere 67 ml di reagente trasfezione a 472 l di media siero ridotti in una provetta. Evitare il contatto del reagente di trasfezione diluito con la parete del tubo. </li><li> Aggiungere 23 mg di pLL3.7 rafforzata proteina fluorescente verde (eGFP) 9 in / ridotto miscela Mezzi di siero di reagenti di trasfezione. </li><li> Incubare la miscela per 20 minuti a RT. </li><li> Sostituire mezzi di crescita delle cellule Caco-2 &#39;con 8 ml di 10% FBS in DMEM. </li><li> Aggiungere il composto DNA / reagente trasfezione / ridotto supporto siero goccia a goccia al piatto. </li><li> Agitare delicatamente i piatti più volte in un avanti e badirezione ckward seguita da una direzione verso sinistra e verso destra. </li><li> Porre la capsula nel C incubatore 37 °. </li><li> Sostituire il supporto nel piatto con 10 ml di terreni di crescita giorno dopo l&#39;avvio l&#39;incubazione. </li><li> Monitorare l&#39;espressione genica. <ol><li> Monitorare l&#39;espressione genica senza 4 &#39;, 6-diamidino-2-fenilindolo (DAPI) colorazione. Utilizzando un microscopio a fluorescenza, determinare la percentuale di cellule che sono verde. La percentuale rappresenta l&#39;efficienza di trasfezione. </li><li> Monitorare l&#39;espressione genica con colorazione DAPI. <ol><li> Lavare le cellule con tampone fosfato salino (PBS) tre volte. </li><li> Fissare le cellule aggiungendo 10 ml di formaldeide al 4% in PBS (10 min di incubazione a RT). </li><li> Lavare le cellule con PBS per tre volte. </li><li> Incubare le cellule al buio per 15 minuti a RT con 8 ml di PBS contenente 1: 500 DAPI, 1% BSA, 0,01% digitonina. </li><li> Lavare le cellule con PBS per tre volte. </li><li> Ucantare un microscopio a fluorescenza, determinare il numero di cellule che sono verde / blu rispetto a quelli che sono blu (Figura 2 pannello superiore). La percentuale calcolata rappresenta l&#39;efficienza di trasfezione. </li></ol></li></ol></li></ol></li><li> Utilizzando il sistema sovraespressione lentivirus 10 <ol><li> Seme umano rene embrionale (HEK) le cellule 293T in un 15 centimetri piatto di coltura tissutale (10% FBS in DMEM come loro mezzi di crescita). Nota: Quando le cellule sono circa il 60-70% confluenti, sono pronti per essere transfettate. </li><li> Aggiungere 162 ml di reagente trasfezione di 1.133 l di media siero ridotti in una provetta 11. Evitare il contatto del reagente di trasfezione diluito con la parete del tubo. </li><li> Aggiungere 24 mg di pLL3.7 eGFP (o un altro costrutto), 15,6 mg di elemento risposta Rev (RRE), 6.0 mg di REV, e 8,4 mg di vescicolare glicoproteina virus della stomatite (VSVG) nel reagente trasfezione / reduced miscela Mezzi di siero. </li><li> Incubare la miscela per 20 minuti a RT. </li><li> Sostituire il supporto di crescita con 16 ml di 10% FBS in DMEM. </li><li> Aggiungere il composto DNA / reagente trasfezione / ridotto supporto siero goccia a goccia al piatto. </li><li> Agitare delicatamente i piatti più volte in avanti e indietro, seguito da una direzione verso sinistra e verso destra. E &#39;normale vedere alcune delle cellule HEK293T distaccati a questo punto. </li><li> Porre la capsula in incubatrice. </li><li> Dopo 24 ore di incubazione, sostituire il supporto nel piatto con 25 ml di terreni di crescita. </li><li> Il giorno seguente, raccogliere i mezzi di crescita contenente il lentivirus (prima raccolta). </li><li> Ripetere il punto 2.2.10 per raccogliere più lentivirus (la seconda raccolta). </li><li> PISCINA La prima e la seconda raccolta, e rimuovere detriti cellulari mediante centrifugazione (2500 xg per 5 min). </li><li> Filtrare la collezione con un filtro a perdere per bottiglia (0,45 micron pori), econcentrare il virus mediante centrifugazione dalla collezione 31.400 xg per 2 ore a temperatura ambiente (JA-20 rotore). Contrassegnare il fondo della provetta in cui si trova il pellet. Decantare il surnatante, e lasciare la provetta capovolta a temperatura ambiente per circa 15 min. </li><li> Risospendere il pellet contenente il virus con 100 ml di PBS 1X. Evitare bolle. </li><li> Conservare il virus concentrato a -80 ° C. Evitare cicli di congelamento-scongelamento. </li><li> Determinare la quantità ottimale di virus necessari per trasdurre cellule Caco-2 mediante titolazione. Per risparmiare sui costi, utilizzare una piastra da 24 pozzetti (0,3 ml di crescita media / pozzo) invece di un piatto di coltura di tessuti 10 centimetri per la titolazione. <ol><li> Aggiungere quantità crescenti del virus concentrati (0, 1, 2, 5, 10, 25, e 50 ml / pozzetto) supplementato con polibrene (concentrazione finale = 5 mg / ml) al 40-70% confluenti cellule Caco-2. Agitare la piastra da 24 pozzetti dolcemente, come descritto al punto 1.1.2. </li><li> Monitorare la loro espressione genica (Fase 2.1.10.) (Figura 2 bottom4 pannelli). </li></ol></li><li> Dal momento che l&#39;espressione del transgene è generalmente sostenuta, di mantenere le cellule trasdotte per futuri esperimenti e monitorare continuamente la loro espressione genica (Step 2.1.10.). </li></ol></li></ol> 3. stimolare la secrezione Lipoprotein <ol><li> Utilizzando la normale piatto di coltura tissutale <ol><li> Preparare una miscela di lipidi 100X mescolando 50 mg di acido oleico, 40 mg di lecitina, e 48 mg di sodio taurocolato. Portare il volume a 900 ml con PBS (sufficiente per 8 piatti). Vortex la miscela lipidica vigorosamente. </li><li> Aggiungere 900 ml di miscela lipidica 100X in 89,1 ml di terreni di crescita. Mescolare e filtrare i media lipidi contenenti. Le concentrazioni finali (1X) di acido oleico, lecitina e sodio taurocolato sono 2,0 mM, 1,36 mM e 1,0 mM, rispettivamente. </li><li> Aggiungere 10 ml di media-lipidico contenente alle cellule. </li><li> Incubare le cellule con i media lipidi contenenti per 4 ore a 37° C incubatore. </li><li> Lavare le cellule con PBS per tre volte. </li><li> Aggiungere 10 ml di mezzi di crescita per le cellule a raccogliere la secrezione delle lipoproteine. </li><li> Incubare per 2 ore in un incubatore a 37 °. </li><li> Raccogliere la media-lipoproteine ​​contenenti. </li></ol></li><li> Utilizzando il sistema di membrana permeabile <ol><li> Seguire i passaggi 3.1.1-3.1.2 per preparare la miscela lipidica. </li><li> Aggiungere 10 ml di media-lipidico contenente alla camera apicale e 10 ml di mezzi di crescita alla camera basolaterale. </li><li> Incubare per 4 ore in un incubatore a 37 °. Raccogliere la media-lipoproteine ​​contenenti nella camera basolaterale. </li></ol></li></ol> 4. Lipoprotein Isolation (Figura 3) <ol><li> Rimuovere i detriti cellulari dal supporto lipoproteine ​​contenenti mediante centrifugazione (2000 xg per 5 min). </li><li> Decantare i mezzi di comunicazione in un tubo da 50 ml. </li><li> Aggiungere 5,95 g di cloruro di sodio ai media. </li><li> Se la sample sarà analizzata dal TEM, aggiungere 1 inibitore della proteasi cocktail della tavoletta per garantire l&#39;integrità delle lipoproteine. </li><li> Portare il volume a 23 ml con acqua (1,2 g / ml NaCl soluzione densità). </li><li> Sciogliere completamente soluti. </li><li> Decantare tutta la soluzione in un tubo di policarbonato ultracentrifuga. </li><li> Sovrapporre delicatamente la soluzione densità di 1,2 g / ml con 0,5 ml di acqua (1,0 g / ml densità). </li><li> Bilanciare il tubo in peso e non in volume. </li><li> Caricare delicatamente i tubi nel rotore T865. </li><li> Spin i campioni nel ultracentrifuga a 429.460 xg per 24 ore a 4 ° C. </li><li> Immediatamente isolare la parte superiore di 0,5 ml di soluzione delicatamente pipettando. Mantenere il tubo più fermi possibile. </li></ol> Analisi 5. TEM <ol><li> Preparare 5 ml di 2% di acido fosfotungstico (w / v) e regolare il pH a 6,0. </li><li> Filtrare l&#39;acido fosfotungstico 2% con un filtro a siringa (dimensione dei pori: 0,2 um). </li><li> Goccia 20 ml di campione lipoproteina su un piatto sterile. </li><li> Goccia 20 ml di 2% di acido fosfotungstico filtrato accanto al campione sullo stesso piatto. </li><li> Cadere delicatamente una griglia EM con il lato opaco appoggiata sul campione. </li><li> Incubare a temperatura ambiente per 1 min. </li><li> Battere delicatamente il lato della griglia EM su una carta da filtro per rimuovere il campione dalla griglia. </li><li> Rilasciare delicatamente la griglia EM con il lato opaco appoggiato sul acido fosfotungstico 2%. </li><li> Incubare a temperatura ambiente per 1 min. </li><li> Battere delicatamente il lato della griglia EM su una carta da filtro per rimuovere l&#39;acido fosfotungstico dalla rete. </li><li> Utilizzando un TEM, catturare le immagini delle lipoproteine ​​(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.

In questo documento, i due sistemi che possono essere utilizzati per mantenere cellule Caco-2 sono descritte, cioè, il piatto di coltura tissutale regolare e la membrana permeabile. I vantaggi di utilizzare il sistema di membrana permeabile includono la separazione del apicale e basolaterale compartimenti, e la capacità di incubare la miscela lipidica e raccogliere la secrezione lipoproteina simultaneamente. Tuttavia, gli inserti membrana permeabile sono costosi, e la loro membrana di policarbonato non consentono una ...

Studi di assorbimento intestinale dei grassi alimentari e farmaci e vitamine liposolubili possono essere condotti in vivo, utilizzando un modello di fistola linfatica 1-4. Tuttavia, le tecniche chirurgiche coinvolte non sono solo una sfida, ma anche costoso. Sebbene in vivo approcci basati sull&#39;analisi fecale possono essere utilizzati, sono utilizzati principalmente per determinare la percentuale di assorbimento dal tratto gastrointestinale 2,5. Il modello in vitro descritto in questo documento è più conveniente, e le tecniche coinvolte sono forse meno impegnativo. Studi modificazione genetica sono anche più economici e quando sono condotti utilizzando questo modello in vitro in termini di tempo di meno. Poiché i materiali liposolubili che sono presi da enterociti sono confezionati in lipoproteine ​​6,7, l&#39;efficacia di questo modello in vitro per produrre lipoproteine ​​è cruciale. Le due intestina principalel lipoproteine ​​sono chilomicroni e molto lipoproteine ​​a bassa densità (VLDL). Chilomicroni, definiti come lipoproteine ​​a 80 nm o più di diametro, sono prodotti rigorosamente dal piccolo intestino quando sono abbondantemente presenti nel lume gastrointestinale lipidi. Dal momento che sono i più grandi lipoproteine, chilomicroni sono plausibilmente i trasportatori lipidici più efficienti. Questo modello in vitro, che è capace di produrre chilomicroni 8, può essere usato per studiare l&#39;assorbimento grassi, liposolubile vitamina assorbimento da parte dell&#39;intestino e biodisponibilità orale lipofilo. La presenza di molecole liposolubili, vitamine o farmaci nella frazione lipoproteina è un indicatore della loro assorbimento da parte dell&#39;intestino tenue. Come discusso in precedenza, questo modello può essere utilizzato per migliorare la biodisponibilità orale farmaco lipofila 6. Questo documento descrive come cellule Caco-2 dovrebbero essere mantenute in membrana permeabile o regolari piastre di coltura dei tessuti, come le mi lipidixture per stimolare la produzione di lipoproteine ​​dovrebbe essere preparato, come il sistema di espressione lentivirus può essere impiegato per realizzare sovraespressione efficace, e come dovrebbe essere analizzato lipoproteine ​​isolate.

<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 visualizza normali 13 giorni post-confluenti cellule Caco-2. La comparsa di strutture a forma di cupola e gocce lipidiche intracellulari sono caratteristici di differenziate cellule Caco-2. Quando le cellule Caco-2 non sono dispersi ugualmente durante la semina, si ciuffo e invadere in alcune zone del piatto; e ci saranno alcune aree nel piatto, senza tutte le cellule. Vorticoso e mettendo il piatto su una superficie inclinata deve essere evitata. E &#39;anche importante notare che il post-conf...

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.

Utilizzando cellule Caco-2 allo Studio Lipid Trasporto dall&#39;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

18.6K Views.  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.

Test di sensibilità visiva alla velocità e direzione del movimento in lucertole

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

Ricerca

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.

Computer-Generated modello animale Stimoli

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

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.

Misurazione di trasporto cationico da Na, K e H, K-ATPasi<em&gt; Xenopus</em&gt; Gli oociti per spettrofotometria di assorbimento atomico: una alternativa a saggi radioisotopi

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.

Metodi per studiare epiteliale Trasporti funzione delle proteine ​​e di espressione in nativo Intestino e Caco-2 cellule coltivate in 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.

Mixed colture primarie di murino tenue finalizzate allo studio di Gut secrezione dell&#39;ormone e imaging cellulare dal vivo di celle enteroendocrine

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.

Membrane lipidiche a doppio strato tethered per monitorare il trasferimento di calore tra nanoparticelle d'oro e membrane lipidiche

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

Isolation of Mouse Interstitial Valve Cells to Study the Calcification of the Aortic Valve In Vitro

The calcification of aortic valve cells is the hallmark of aortic stenosis and is associated with valve cusp fibrosis. Valve interstitial cells (VICs) play an important role in the calcification process in aortic stenosis through the activation of their dedifferentiation program to osteoblast-like cells. Mouse VICs are a good in vitro tool for the elucidation of the signaling pathways driving the mineralization of the aortic valve cell. The method described herein, successfully used by these authors, explains how to obtain freshly isolated cells. A two-step collagenase procedure was performed with 1 mg/mL and 4.5 mg/mL. The first step is crucial to remove the endothelial cell layer and avoid any contamination. The second collagenase incubation is to facilitate the migration of VICs from the tissue to the plate. In addition, an immunofluorescence staining procedure for the phenotype characterization of the isolated mouse valve cells is discussed. Furthermore, the calcification assay was performed in vitro by using the calcium reagent measurement procedure and alizarin red staining. The use of mouse valve cell primary culture is essential for testing new pharmacological targets to inhibit cell mineralization in vitro.

Isolation of Mouse Interstitial Valve Cells to Study the Calcification of the Aortic Valve In Vitro

This article describes the isolation of mouse aortic valve cells by a two-step collagenase procedure. Isolated mouse valve cells are important for performing different assays, such as this in vitro calcification assay, and for investigating the molecular pathways leading to aortic valve mineralization.

Isolamento delle celle della valvola interstiziale del topo per studiare la calcificazione della valvola aortica in vitro

The calcification of aortic valve cells is the hallmark of aortic stenosis and is associated with valve cusp fibrosis. Valve interstitial cells (VICs) ...

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.

Il saggio biologico delle cellule Caco-2 per la misurazione della biodisponibilità del ferro negli alimenti

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.

Preparazione del campione per l'analisi rapida dei lipidi nel cervello della Drosophila utilizzando la spettrometria di massa a desorbimento/ionizzazione laser assistita da matrice

Lipid profiling, or lipidomics, is a well-established technique used to study the entire lipid content of a cell or tissue. Information acquired ...

Studio del meccanismo di trasporto dello zinco nelle cellule di mammifero con il saggio FluoZin-3

Characterizing Mammalian Zinc Transporters Using an In Vitro Zinc Transport Assay

Transition metals such as Zn2+ ions must be tightly regulated due to their cellular toxicity. Previously, the activity of Zn2+ transporters was measured indirectly by determining the expression level of the transporter under different concentrations of Zn2+. This was done by utilizing immunohistochemistry, measuring mRNA in the tissue, or determining the cellular Zn2+ levels. With the development of intracellular Zn2+ sensors, the activities of zinc transporters are currently primarily determined by correlating changes in intracellular Zn2+, detected using fluorescent probes, with the expression of the Zn2+ transporters. However, even today, only a few labs monitor dynamic changes in intracellular Zn2+ and use it to measure the activity of zinc transporters directly. Part of the problem is that out of the 10 zinc transporters of the ZnT family, except for ZnT10 (transports manganese), only zinc transporter 1 (ZnT1) is localized at the plasma membrane. Therefore, linking the transport activity to changes in the intracellular Zn2+ concentration is hard. This article describes a direct way to determine the zinc transport kinetics using an assay based on a zinc-specific fluorescent dye, FluoZin-3. This dye is loaded into mammalian cells in its ester form and then trapped in the cytosol due to cellular di-esterase activity. The cells are loaded with Zn2+ by utilizing the Zn2+ ionophore pyrithione. The ZnT1 activity is assessed from the linear part of the reduction in fluorescence following the cell washout. The fluorescence measured at an excitation of 470 nm and emission of 520 nm is proportional to the free intracellular Zn2+. Selecting the cells expressing ZnT1 tagged with the mCherry fluorophore allows for monitoring only the cells expressing the transporter. This assay is used to investigate the contribution of different domains of ZnT1 protein to the transport mechanism of human ZnT1, a eukaryotic transmembrane protein that extrudes excess zinc from the cell.

Autore in primo piano: Esplorare la regolazione cellulare dello zinco attraverso la funzionalità ZnT1 

Zinc transport has proven challenging to measure due to the weak causal links to protein function and the low temporal resolution. This protocol describes a method for monitoring, with high temporal resolution, Zn2+ extrusion from living cells by utilizing a Zn2+ sensitive fluorescent dye, thus providing a direct measure of Zn2+ efflux.

Caratterizzazione dei trasportatori di zinco nei mammiferi mediante un saggio di trasporto dello zinco in vitro

Transition metals such as Zn2+ ions must be tightly regulated due to their cellular toxicity. Previously, the activity of Zn2+ transporters was measured ...