Name: methods to study epithelial transport protein function and expression in native intestine and caco-2 cells grown in 3d
Uploaded: 2017-03-16T13:00:00
Description: Watch this Scientific Journal Video about Methods to Study Epithelial Transport Protein Function and Expression in Native Intestine and Caco-2 Cells Grown in 3D at JoVE.com

We thank Mr. Christopher Manzella, MD/PhD student in the RKG laboratory, for helping to edit and proofread our manuscript.

1. Study of Intestinal Transporters in a 3D Culture System of Caco-2: Growing 3D Caco-2 Cell Culture on a Gelatinous Protein Mixture
<ol>
	<li>Thaw growth factor reduced gelatinous protein mixture on ice for 8 h (or O/N) at 4 &#176;C. Once thawed, make 1 mL or 500 &#181;L aliquots for use or store at -20 &#176;C for later use.</li>
	<li>On the day of culture, precool the culture plates (for RNA and protein extraction) or chambered slides (for immunostaining) on ice. Add 30 &#181;L of gelatinous protein mixture to each ...

<ol>
	<li>Simon-Assmann, P., Turck, N., Sidhoum-Jenny, M., Gradwohl, G., Kedinger, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+models+of+intestinal+epithelial+cell+differentiation.">In vitro models of intestinal epithelial cell differentiation.</a> Cell Biol Toxicol. 23 (4), 241-256 (2007).</li><li>Shen, C., Meng, Q., Zhang, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+of+3D+printed+insert+for+hanging+culture+of+Caco-2+cells.">Design of 3D printed insert for hanging culture of Caco-2 cells.</a> Biofabrication. 7 (1), 015003 (2014).</li><li>Jaffe, A. B., Kaji, N., Durgan, J., Hall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cdc42+controls+spindle+orientation+to+position+the+apical+surface+during+epithelial+morphogenesis.">Cdc42 controls spindle orientation to position the apical surface during epithelial morphogenesis.</a> J Cell Biol. 183 (4), 625-633 (2008).</li><li>Martel, F., Monteiro, R., Lemos, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uptake+of+serotonin+at+the+apical+and+basolateral+membranes+of+human+intestinal+epithelial+(Caco-2)+cells+occurs+through+the+neuronal+serotonin+transporter+(SERT).">Uptake of serotonin at the apical and basolateral membranes of human intestinal epithelial (Caco-2) cells occurs through the neuronal serotonin transporter (SERT).</a> J Pharmacol Exp Ther. 306 (1), 355-362 (2003).</li><li>Gill, R. K., 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=Function,+expression,+and+characterization+of+the+serotonin+transporter+in+the+native+human+intestine.">Function, expression, and characterization of the serotonin transporter in the native human intestine.</a> Am J Physiol Gastrointest Liver Physiol. 294 (1), G254-G262 (2008).</li><li>Esmaili, 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=Enteropathogenic+Escherichia+coli+infection+inhibits+intestinal+serotonin+transporter+function+and+expression.">Enteropathogenic Escherichia coli infection inhibits intestinal serotonin transporter function and expression.</a> Gastroenterology. 137 (6), 2074-2083 (2009).</li><li>Wade, P. R., 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=Localization+and+function+of+a+5-HT+transporter+in+crypt+epithelia+of+the+gastrointestinal+tract.">Localization and function of a 5-HT transporter in crypt epithelia of the gastrointestinal tract.</a> J Neurosci. 16 (7), 2352-2364 (1996).</li><li>Clarke, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+to+Ussing+chamber+studies+of+mouse+intestine.">A guide to Ussing chamber studies of mouse intestine.</a> Am J Physiol Gastrointest Liver Physiol. 296 (6), G1151-G1166 (2009).</li><li>Debnath, J., Muthuswamy, S. K., Brugge, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphogenesis+and+oncogenesis+of+MCF-10A+mammary+epithelial+acini+grown+in+three-dimensional+basement+membrane+cultures.">Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures.</a> Methods. 30 (3), 256-268 (2003).</li><li>Vedeler, A., Pryme, I. F., Hesketh, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin+induces+changes+in+the+subcellular+distribution+of+actin+and+5'-nucleotidase.">Insulin induces changes in the subcellular distribution of actin and 5'-nucleotidase.</a> Mol Cell Biochem. 108 (1), 67-74 (1991).</li><li>Botvinkin, A. D., Selimov, M. A., Zgurskaia, G. N., Kulikov, L. G., Aksenova, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=[Detection+of+rabies+virus+antibodies+in+human+blood+serum+by+an+immunoenzyme+method].">[Detection of rabies virus antibodies in human blood serum by an immunoenzyme method].</a> Vopr Virusol. 31 (3), 333-334 (1986).</li><li>Woods, G. L., Mills, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+dexamethasone+on+detection+of+herpes+simplex+virus+in+clinical+specimens+by+conventional+cell+culture+and+rapid+24-well+plate+centrifugation.">Effect of dexamethasone on detection of herpes simplex virus in clinical specimens by conventional cell culture and rapid 24-well plate centrifugation.</a> J Clin Microbiol. 26 (6), 1233-1235 (1988).</li><li>Chase, A. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acyclovir+for+shingles.">Acyclovir for shingles.</a> Br Med J (Clin Res Ed. 295 (6603), 926-927 (1987).</li><li>Churchill, P. F., Churchill, S. A., Martin, M. V., Guengerich, F. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+rat+liver+cytochrome+P-450UT-H+cDNA+clone+and+comparison+of+mRNA+levels+with+catalytic+activity.">Characterization of a rat liver cytochrome P-450UT-H cDNA clone and comparison of mRNA levels with catalytic activity.</a> Mol Pharmacol. 31 (2), 152-158 (1987).</li><li>Steiner, M., Tateishi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+and+transport+of+calcium+in+human+platelets.">Distribution and transport of calcium in human platelets.</a> Biochim Biophys Acta. 367 (2), 232-246 (1974).</li><li>McClelland, M., Nelson, M., Cantor, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+Mbo+II+methylase+(GAAGmA)+from+Moraxella+bovis:+site+specific+cleavage+of+DNA+at+nine+and+ten+base+pair+sequences.">Purification of Mbo II methylase (GAAGmA) from Moraxella bovis: site specific cleavage of DNA at nine and ten base pair sequences.</a> Nucleic Acids Res. 13 (20), 7171-7182 (1985).</li><li>Chatterjee, I. s. h. i. t. a., K, A., Gill, R. a. v. i. n. d. e. r. . K., Alrefai, W. a. d. d. a. h. . A., Dudeja, P. r. a. d. e. e. p. . K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+Cell+Culture+of+CaCo2:+A+Better+Model+to+Study+the+Functionality+and+Regulation+of+Intestinal+Ion+Transporters.">3D Cell Culture of CaCo2: A Better Model to Study the Functionality and Regulation of Intestinal Ion Transporters.</a> Gastroenterology. 146 (5, Supplement 1), S-654 (2014).</li><li>Fagg, R. H., Hyslop, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+a+variant+strain+of+foot-and-mouth+disease+virus+(type+O)+during+passage+in+partly+immunized+cattle.">Isolation of a variant strain of foot-and-mouth disease virus (type O) during passage in partly immunized cattle.</a> J Hyg (Lond). 64 (4), 397-404 (1966).</li><li>Shilkina, N. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=[The+aspects+of+the+problem+of+systemic+vasculitis+open+to+discussion]).">[The aspects of the problem of systemic vasculitis open to discussion]).</a> Ter Arkh. 61 (12), 102-106 (1989).</li><li>Derichs, N., 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=Intestinal+current+measurement+for+diagnostic+classification+of+patients+with+questionable+cystic+fibrosis:+validation+and+reference+data.">Intestinal current measurement for diagnostic classification of patients with questionable cystic fibrosis: validation and reference data.</a> Thorax. 65 (7), 594-599 (2010).</li></ol>

Fully differentiated Caco-2 cell monolayers have been used extensively as polarized epithelial monolayers to study intestinal transport10,11,12,13,14,15. However, to mimic the physiological organization of intestinal epithelial cells, there has been considerable interest in the development of a Caco-2 culture in 3D. A number ...

A correction was made to the Authors section in: <a href="http://www.jove.com/video/55304">Methods to Study Epithelial Transport Protein Function and Expression in Native Intestine and Caco-2 Cells Grown in 3D</a>.
One of the authors&#39; names was corrected from:
Arivarasu N. Anabazhagan
to:
Arivarasu N. Anbazhagan

A correction was made to the Authors section in: <a href="http://www.jove.com/video/55304">Methods to Study Epithelial Transport Protein Function and Expression in Native Intestine and Caco-2 Cells Grown in 3D</a>.

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

The intestinal epithelium is equipped with various transport proteins (channels, ATPases, co-transporters and exchangers) that perform numerous functions ranging from the absorption of nutrients, electrolytes, and drugs to the secretion of fluid and ions in the lumen. Transport proteins generate electrochemical gradients that permit the movement of ions or molecules in a vectorial manner. This is achieved by the asymmetric distributions of the transport systems in the apical and basolateral membranes of polarized epithelial cells. In addition, tight junctions, which tether adjacent epithelial cells, play an important role in this process by serving as a barrier to the intramembrane diffusion of components between the apical and basolateral membrane domains. Appropriate model systems mimicking these characteristic features of the native intestine (i.e.&#160;polarity, differentiation, and tight junction integrity) are critical for the study of the functionality of epithelial transport systems.
With respect to models, the typical cell lines used currently in intestinal epithelial transport research are Caco-2, a model of fully differentiated, absorptive small intestinal epithelial cells; and T84 cells or HT-29 subclones, models of crypt-derived large intestinal epithelial cells1. Conventionally, these cell lines are grown as monolayers in plastic surfaces or in coated transwell inserts. Trans-well cell culture inserts to some extent resemble the in vivo environment by allowing polarized cells to feed basolaterally. However, a limitation in conventional 2D&#160;culture systems is that the cells are forced to adapt to an artificial, flat, and rigid surface. Thus, the physiological complexity of the native epithelia is not accurately reflected in a 2D system. This limitation has been overcome by methods to grow cells in 3D&#160;in a specific microenvironment, such as gelatinous protein mixture, containing a variety of extracellular matrix components2,3. Nevertheless, the in vitro cultures cannot simulate the complexity of the intestinal epithelium, which has multiple cell types and region-specific architecture in the intestine. Thus, the studies using cell culture models require further validation in the native intestine. Using the in vitro 3D cell culture and mouse intestinal mucosa, we describe here simple methods to study the regulation of the intestinal serotonin transporter (SLC6A4, SERT).
The SERT transporter regulates the extracellular availability of an important hormone and neurotransmitter, 5-hydroxytryptamine (5-HT), by rapidly transporting it through a Na+/Cl--dependent process. SERT is a known target of anti-depressants and has recently emerged as a novel therapeutic target of GI disorders, such as diarrhea and intestinal inflammation. Methods to investigate 5-HT uptake in intestinal epithelial cells have been previously described. For example, Caco-2 cells grown on plastic supports or permeable inserts have been shown to exhibit fluoxetine-sensitive 3H-5-HT uptake at both apical and basolateral domains4. The measurement of SERT function in isolated Brush Border Membrane Vesicles (BBMVs) prepared from human organ donor small intestines has also been described by us5. 3H-5-HT uptake in human BBVMs was shown to be fluoxetine-sensitive and Na+/Cl--dependent, and it exhibited saturation kinetics with a Km of 300 nM5. Utilizing similar methods, we also previously measured SERT function as 3H-5-HT uptake in mouse intestinal BBMVs6. However, the preparation of pure plasma membrane vesicles requires a large amount of tissue mucosa. Other methods, such as the radiographic visualization of 3H-5-HT uptake sites, have also been shown previously in guinea pig and rat small intestines7.
The Ussing chamber provides a more physiological system to measure the transport of ions, nutrients, and drugs across various epithelial tissues. The main advantage of the Ussing chamber technique is that it enables the precise measurement of the electrical and transport parameters of intact, polarized intestinal epithelium. Further, to minimize the influence of the intrinsic neuromuscular system, seromuscular stripping of intestinal mucosa can be performed to investigate the regulation of transporters in the epithelium8.
We demonstrate that Caco-2 cells grown in 3D on gelatinous protein form a hollow lumen expressing distinct apical and basolateral markers. These cells show higher expression of SERT than 2D Caco-2 cells. Methods to grow 3D cells and to perform immunostaining or RNA and protein extraction are described. In addition, we describe methods to study SERT function and regulation by TGF-&#946;1, a pleiotropic cytokine, in small intestinal mucosa by utilizing an Ussing chamber technique.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10X PBS</td>
			<td>ATCC</td>
			<td>ATCC&#174; HTB&#173;37&#8482;</td>
			<td>Cells</td>
		</tr>
		<tr>
			<td>10X PBS</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td>Microsope for confocal Imaging</td>
		</tr>
		<tr>
			<td>3[H]-5-HT&#160;</td>
			<td>LabtekII</td>
			<td>152453</td>
			<td>Culturing cells for microscopy</td>
		</tr>
		<tr>
			<td>5-HT&#160;</td>
			<td>Gibco</td>
			<td>70013-032</td>
			<td>Not a hazardous substance</td>
		</tr>
		<tr>
			<td>95%O2- 5%Co2 cylinder</td>
			<td>KPL</td>
			<td>71-00-27</td>
			<td>Not a hazardous substance</td>
		</tr>
		<tr>
			<td>Agar (for Agar plates)</td>
			<td>Fischer</td>
			<td>BP151-100</td>
			<td>Acute toxicity, Ora</td>
		</tr>
		<tr>
			<td>Agar (for electrode)</td>
			<td>Sigma</td>
			<td>G7126-1KG</td>
			<td>Not a hazardous substance</td>
		</tr>
		<tr>
			<td>Alexa FluorPhalloidin</td>
			<td>Sigma</td>
			<td>C3867-1VL</td>
			<td>Not a hazardous substance</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>P6148-500G</td>
			<td>Harmful if swallowed or if inhaled</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>LifeTechnologies</td>
			<td>A12380</td>
			<td>Not a hazardous substance</td>
		</tr>
		<tr>
			<td>CaCo2 Cells</td>
			<td>Sigma</td>
			<td>A8806-5G</td>
			<td>Not a hazardous substance</td>
		</tr>
		<tr>
			<td>Cell lysis Buffer&#160;</td>
			<td>Fischer</td>
			<td>BP337-100</td>
			<td>Not a hazardous substance</td>
		</tr>
		<tr>
			<td>Chabered slides</td>
			<td>Sigma</td>
			<td>S5886-1KG</td>
			<td>Not a hazardous substance</td>
		</tr>
		<tr>
			<td>Collagen Type-1Solution</td>
			<td>Sigma</td>
			<td>S8045-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrodes</td>
			<td>Sigma</td>
			<td>S-0876</td>
			<td></td>
		</tr>
		<tr>
			<td>EMEM</td>
			<td>Gibco by Life Technologies</td>
			<td>25200-056</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Fetal bovine serum</td>
			<td>Corning</td>
			<td>356234</td>
			<td>Used as Extracellular matrix</td>
		</tr>
		<tr>
			<td>Fluoxetine</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Gentamicin</td>
			<td>ATCC</td>
			<td>30-2003</td>
			<td>Cell culture Media</td>
		</tr>
		<tr>
			<td>Glycin</td>
			<td>Cell Signaling, Danvers, MA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hepes&#160;</td>
			<td>Roche</td>
			<td>11836145001</td>
			<td></td>
		</tr>
		<tr>
			<td>Indomethacin</td>
			<td>Gibco by Life Technologies</td>
			<td>15070-063</td>
			<td></td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td>Gibco by Life Technologies</td>
			<td>15710-064</td>
			<td></td>
		</tr>
		<tr>
			<td>KOH</td>
			<td>Gibco by Life Technologies</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr>
			<td>L-ascorbic acid&#160;</td>
			<td>Gibco by Life Technologies</td>
			<td>10082147</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid scintillation counter&#160;</td>
			<td>Gibco by Life Technologies</td>
			<td>70013--032</td>
			<td></td>
		</tr>
		<tr>
			<td>LSM710 Meta</td>
			<td>Physiologic Instruments, San Diego, CA&#160;</td>
			<td>EM-CSYS-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Mannitol</td>
			<td>Physiologic Instruments, San Diego, CA&#160;</td>
			<td>P2304</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Physiologic Instruments, San Diego, CA&#160;</td>
			<td>VCC MC8</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Physiologic Instruments, San Diego, CA&#160;</td>
			<td>DM MC6</td>
			<td></td>
		</tr>
		<tr>
			<td>Multichannel Voltage/current Clamp</td>
			<td>Physiologic Instruments, San Diego, CA&#160;</td>
			<td>P2300</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Physiologic Instruments, San Diego, CA&#160;</td>
			<td>P2023-100</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher Scientific, Hampton, NH</td>
			<td>BP1423</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma-Aldrich, St Louis, MO</td>
			<td>S5886</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Goat Serum (10%)</td>
			<td>Fisher Scientific, Hampton, NH</td>
			<td>S233</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich, St Louis, MO</td>
			<td>P288</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen-Strp</td>
			<td>Sigma-Aldrich, St Louis, MO</td>
			<td>C3881</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein assay reagent</td>
			<td>Sigma-Aldrich, St Louis, MO</td>
			<td>10420</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase Inhibitor Cocktail</td>
			<td>MEDOX, Chicago, IL&#160;</td>
			<td>style G- 12300</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA easy Mini kit</td>
			<td>Sigma-Aldrich, St Louis, MO</td>
			<td>M4125</td>
			<td></td>
		</tr>
		<tr>
			<td>Single Channel Electrode Input Module&#160;</td>
			<td>Sigma-Aldrich, St Louis, MO</td>
			<td>A92902</td>
			<td></td>
		</tr>
		<tr>
			<td>Sliders for snapwell Chambers</td>
			<td>Sigma-Aldrich, St Louis, MO</td>
			<td>H9523</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosphate (Na2HPO4)</td>
			<td>Sigma-Aldrich, St Louis, MO</td>
			<td>F132</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium-Hydroxide (NaOH)</td>
			<td>Sigma-Aldrich, St Louis, MO</td>
			<td>T7039</td>
			<td></td>
		</tr>
		<tr>
			<td>TGF-&#946;1</td>
			<td>Perkin-Elmer,Waltham, MA</td>
			<td>NFT1167250UC</td>
			<td></td>
		</tr>
		<tr>
			<td>TritonX-100</td>
			<td>Packard Instruments, Downers Grove, IL</td>
			<td>B1600</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin EDTA</td>
			<td>Sigma-Aldrich, St Louis, MO</td>
			<td>221473</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Bio Rad Laboratories, Hercules, CA</td>
			<td>5000006</td>
			<td></td>
		</tr>
		<tr>
			<td>Ussing Chamber (chamber only)</td>
			<td>Sigma-Aldrich, St Louis, MO</td>
			<td>I7378</td>
			<td></td>
		</tr>
		<tr>
			<td>Ussing Chamber Systems</td>
			<td>Sigma-Aldrich, St Louis, MO</td>
			<td>A1296</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Immunostaining in 3D Caco-2 cysts is shown in Figure 1. A representative image of an XY-plane showing well-demarcated lumen in the 3D cyst stained with phalloidin actin is depicted in Figure 1A. The side facing the lumen denotes the apical side. Epithelial cells cultured in a 3D environment result in a robust epithelial phenotype. Different Caco-2 cysts on day 12, when actin and SERT were co-stained, are shown in Figure 1B. In this repres...

Watch this Scientific Journal Video about Methods to Study Epithelial Transport Protein Function and Expression in Native Intestine and Caco-2 Cells Grown in 3D at JoVE.com

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

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.

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

The overall goal of this protocol is to grow intestinal Caco-2 cells in three dimensions and to demonstrate the use of the Ussing chamber in studies on serotonin transporter regulation. The methods shown here can answer key questions in epithelial transport field, such as how intestinal serotonin transporter, SERT is regulated. The main advantage of the 3D Caco-2 is that they reflect cell-to-cell and cell-to-extracellular matrix interactions more accurately than monolayers.And Ussing chamber technique enables precise measurements of transport function in the intestinal epithelium. The implication of 3D, the Caco cultures extends toward discovery of therapeutics because these models enable screening for agents against diseases associated with altered transport function. Though this method provides insight into serotonin transporter regulation, it can also be applied to studies of other electrolyte transporters such as sodium and chloride.Visual demonstration of these techniques is critical, as stripping the seromuscular layer and mounting the intestinal mucosa in Ussing chambers are difficult to learn. Demonstrating the 3D Caco-2 cell procedures will be Ishita Chatterjee, instructor, and Anoop Kumar, a postdoc fellow from our group. Demonstrating the stripping of the seromuscular layer from mouse ileum will be Sangeeta Tyagi, a senior research specialist from my lab.Also demonstrating the mounting of stripped mucosa and inserting them in Ussing chambers will be Shubha Priyamvada and Arivarasu Natarajan, instructors from my lab. To begin, thaw the growth factor reduced gelatinous protein solution overnight on ice in a four degree Celsius refrigerator. Once thawed, prepare one milliliter, or 500 microliter aliquots.On the day of culture, precool the chambered slides on ice. Then, add 30 microliters of the gelatinous protein solution to each well of the eight-well chambered glass slide and spread it evenly. While plating the gelatinous mixture in the chamber slide or plates, care should be taken to avoid bubble formation as the cells may detach.Place the cultured dish inside a 37 degree Celsius cell culture incubator for 15 to 30 minutes to allow the solution to solidify. Next, using trypsin, detach confluent Caco-2 cells from a culture flask. Then, count the cells in a hemocytometer and centrifuge them at 500 times g at four degrees Celsius for five minutes.Re-suspend the resulting cell pellet in 3D Caco-2 medium to obtain the suspension of the desired density. Using the prepared the cell suspension, seed 4, 000 cells per well onto the glass chamber slides and allow them to grow for 12 to 14 days in a 5%carbon dioxide humidified incubator at 37 degrees Celsius. To stain the cells, first aspirate the medium and fix the cells with 400 microliters of 2%PFA.Continue fixing the cells for 20 minutes at room temperature. After washing the cells twice in PBS, permeabilize them with 0.5%Triton solution in PBS for no longer than 15 minutes. Rinse the cells twice in PBS glycine buffer and then wash the permeabilized cells in IF buffer for ten minutes at room temperature.Block the cells using 5%normal goat serum in IF buffer. Then, incubate the cells with 200 microliters of primary antibody diluted in IF buffer supplemented with 1%goat serum for one to two hours at room temperature. After incubation, wash cells thrice with IF buffer.Incubate washed cells with 200 microliters of secondary antibody diluted in IF buffer supplemented with 1%goat serum for one hour at room temperature. Wash cells with IF buffer thrice for ten minutes. To detach the chamber for the glass slide, first place the slide in the slide base holder, then slide the white lifter through the holder until it contacts the edge of the wells.Gently pull the chamber to remove it from the slide. Use a covered black box to protect the slides from light. Mount the slides with slow anti-fade medium and cover them with cover slips.After allowing the slides to dry for ten minutes at room temperature, seal them with nail polish prior to imaging. Isolate mouse ileum following the procedure described in the text protocol. Then, using scissors, open the intestine longitudinally.Incubate the resulting tissue section in ice-cold gassing KBR buffer containing one micromolar indomethacin for ten minutes. Pin an approximately one centimeter long intestinal section, mucosal side down, to a plate containing 0.5 centimeter thick 7%agarose or cured silicone elastomer. Using a dissection stereo microscope with bottom illumination, strip the seromuscular layers.Then, cut the seromuscular layer with a feather scalpel blade. Use fine forceps to lift the edge of the layer along the longitudinal axis of the intestine. Finally, mount the mucosa carefully on the pins of the slider.Hold the stripped mucosal tissue by the edges to avoid tearing. While mounting the stripped ilial mucosa on pins of the slider, precautions should be taken to prevent tearing the tissue at the pinheads and to minimize damaging the tissue's edges. Prior to tissue treatment, prepare Krebs solution gassed with 95%oxygen, 5%carbon dioxide.Then, pour the solution into a Ussing chamber. Add 10 micromolars glucose as the energy substrate to the serosal bath and 10 micromolar mannitol for maintaining osmotic balance to the mucosal bath. Subsequently, insert the slider with the mounted mucosa into the chamber to expose both the apical and the basolateral sides of the tissue to the Krebs solution.Equilibrate the ileal tissue in the bath for ten minutes. Then, pretreat the apical side with 10 micromolar fluoxetine for 30 minutes for the measurement of the mucosal to serosal flux. Ultimately, treat the basolateral side of the tissue with 10 nanograms per milliliter TGF beta 1 for one hour, and then incubate the apical side with 20 nanomolar tritiated 5-HT for 30 minutes.To calculate mucosal to serosal flux rates, collect 0.75 milliliter aliquots from the serosal reservoir, replacing them with identical volumes of the bath medium to prevent hydrostatic pressure differences across the mucosa. To quantify the tritiated 5-HT accumulated in the tissue, remove the mucosa from the slider. Wash it once with ice-cold KRB buffer and place it in a glass culture tube.Incubate the mucosa in 0.5 milliliters of 10%KOH overnight at 37 degrees Celsius to lyse the tissue. Then, measure the radioactivity in 150 microliter aliquots of the lysates in triplicate using a liquid scintillation counter. Using the Bradford method, measure the protein concentration in three to five microliter aliquots of the lysates.Shown here are Caco-2 cysts grown in 3D culture stained for actin and Caco-2 3D cysts stained simultaneously for actin, nuclei and SERT protein. SERT is visible in the luminal membrane and in subapical compartments. To further confirm the advantage of Caco-2 3D spheres over 2D Caco-2 monolayers, western blot analysis of SERT protein expression was performed.The results showed the enhanced expression of SERT protein in Caco-2 3D spheres compared to 2D Caco-2 cells. Finally, a Ussing chamber system was used to show the TGF beta increases mucosal to serosal flux, reflecting increased 5-HT uptake from the luminal membrane and enhanced 5-HT accumulation observed in the ileal mucosa. Such findings are supported by the observed sensitivity of 5-HT uptake to fluoxetine treatment that corresponds to SERT expression on the luminal membrane.Once mastered, this technique of stripping and mounting the ileal mucosa can be completed in 15 minutes. While adopting this procedure, it's important to remember that upon removal from the animal, the extravio intestinal preparation has limited viability and it may last for up to three hours. 3D Caco-2 cysts could be utilized for various studies such as membrane trafficking events, gene expression, or protein-protein interaction of epithelial transporters.After its development, the 3D Caco-2 technique paves the way for researchers in the field of intestinal transport to explore fluid movement and to study the pathophysiology of darial diseases. Do not forget that working with radioactivity can be extremely hazardous, and precautions, such as the use of PPE and proper decontamination procedures should always be taken. After watching this video, you will be able to grow Caco-2 cells in 3D cultures and to utilize these cultures to investigate epithelial transporter expression.You will also be able to utilize the Ussing chamber to study serotonin transport function in the native mouse intestine.

methods-to-study-epithelial-transport-protein-function-expression

Research

JoVE Journal

Biology

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Primary Human Bronchial Epithelial Cells Grown from Explants

Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the function and structure of human epithelial cells. Here we describe in detail the method of growing bronchial epithelial cells from bronchial airway tissue that is harvested by the surgeon at the times of lung surgery (e.g. lung cancer or lung volume reduction surgery). With ethics approval and informed consent, the surgeon takes what is needed for pathology and provides us with a bronchial portion that is remote from the diseased areas. The tissue is then used as a source of explants that can be used for growing primary bronchial epithelial cells in culture. Bronchial segments about 0.5-1cm long and &le;1cm in diameter are rinsed with cold EBSS and excess parenchymal tissue is removed. Segments are cut open and minced into 2-3mm3 pieces of tissue. The pieces are used as a source of primary cells. After coating 100mm culture plates for 1-2 hr with a combination of collagen (30 &mu;g/ml), fibronectin (10 &mu;g/ml), and BSA (10 &mu;g/ml), the plates are scratched in 4-5 areas and tissue pieces are placed in the scratched areas, then culture medium (DMEM/Ham F-12 with additives) suitable for epithelial cell growth is added and plates are placed in an incubator at 37&deg;C in 5% CO2 humidified air. The culture medium is changed every 3-4 days. The epithelial cells grow from the pieces forming about 1.5 cm diameter rings in 3-4 weeks. Explants can be re-used up to 6 times by moving them into new pre-coated plates. Cells are lifted using trypsin/EDTA, pooled, counted, and re-plated in T75 Cell Bind flasks to increase their numbers. T75 flasks seeded with 2-3 million cells grow to 80% confluence in 4 weeks. Expanded primary human epithelial cells can be cultured and allowed to differentiate on air-liquid interface. Methods described here provide an abundant source of human bronchial epithelial cells from freshly isolated tissues and allow for studying these cells as models of disease and for pharmacology and toxicology screening.

Here we describe a detailed method for growing primary human bronchial epithelial cells from explants of human bronchial airway tissue including differentiated growth on an air-liquid interface. This method provides an abundant source of primary cells for investigating the role of the airway epithelium in human lung health and disease.

Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the ...

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression both in terms of tissue distribution and developmental stage. Protocols are described for the use of WISH of zebrafish embryos using antisense RNA probes labeled with digoxigenin. Probes are generated by incorporating digoxigenin-linked nucleotides through in vitro transcription of gene templates that have been cloned and linearized. The chorions of embryos harvested at defined developmental stages are removed before incubation with specific probes. Following a washing procedure to remove excess probe, embryos are incubated with anti-digoxigenin antibody conjugated with alkaline phosphatase. By employing a chromogenic substrate for alkaline phosphatase, specific gene expression can be assessed. Depending on the level of gene expression the entire procedure can be completed within 2-3 days.

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

The zebrafish, a small tropical fish, has become a popular model for studying gene function during vertebrate development and disease. The temporal and spatial expression of target genes can be determined by in situ hybridization. Our improved protocol allows for the detection of low abundant transcripts with low non-specific background signal.

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

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).

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.

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

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...