Name: using caco-2 cells to study lipid transport by the intestine
Uploaded: 2015-08-20T14:00:00
Description: Watch this Scientific Journal Video about Using Caco-2 Cells to Study Lipid Transport by the Intestine at JoVE.com

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. Maintenance of the Caco-2 Cells
<ol>
	<li>Using regular tissue culture dishes
	<ol>
		<li>Thaw the Caco-2 cells from a frozen vial by placing the vial in a 37 &#176;C water bath, and immediately add them to a 10 cm tissue culture dish containing 10 ml&#160;of pre-warmed growth media (15% fetal bovine serum (FBS) in Dulbecco&#8217;s Modified Eagle Medium (DMEM)).
		<ol>
			<li>When the Caco-2 cells have reached 50-70% confluence, split them 1:6 by incubating the cells with 3 ml&#160;of 0.05% Trypsin / 0.53 mM ethylen...

<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>

In this paper, the two systems that can be used to maintain Caco-2 cells are described, namely, the regular tissue culture dish and the permeable membrane. The benefits of using the permeable membrane system include the separation of the apical and the basolateral compartments, and the ability to incubate the lipid mixture and collect the lipoprotein secretion simultaneously. However, the permeable membrane inserts are expensive, and their polycarbonate membrane does not allow for good cell visibility. One of the advanta...

Studies of intestinal absorption of dietary fat, and lipid-soluble drugs and vitamins can be conducted in vivo by using a lymph fistula model 1&#8211;4. However, the surgical techniques involved are not only challenging, but also costly. Although in vivo approaches based on fecal analysis may be utilized, they are used mainly to determine the percent uptake by the gastrointestinal tract 2,5. The in vitro model described in this paper is more cost effective, and the techniques involved are arguably less challenging. Genetic modification studies are also more economical and less time-consuming when they are conducted using this in vitro model.
Since lipid-soluble materials that are taken up by enterocytes are packaged into lipoproteins 6,7, the effectiveness of this in vitro model to produce lipoproteins is crucial. The two main intestinal lipoproteins are chylomicrons and very low-density lipoproteins (VLDL). Chylomicrons, defined as lipoproteins with 80 nm or more in diameter, are produced strictly by the small intestine when lipids are abundantly present in the gastrointestinal lumen. Since they are the largest lipoproteins, chylomicrons are conceivably the most efficient lipid transporters. This in vitro model, which is capable of producing chylomicrons 8, can be used to study dietary fat absorption, lipid-soluble vitamin absorption by the gut, and oral lipophilic drug bioavailability. The presence of lipid-soluble molecules, vitamins, or drugs in the lipoprotein fraction is an indicator of their absorption by the small intestine. As previously discussed, this model can be used to improve oral lipophilic drug bioavailability 6.
This paper describes how Caco-2 cells should be maintained in permeable membrane or regular tissue culture dishes, how the lipid mixture for stimulating lipoprotein production should be prepared, how the lentivirus expression system can be employed to achieve effective overexpression, and how the isolated lipoproteins should be analyzed.

<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).

Figure 1 displays normal 13-days post-confluent Caco-2 cells. The appearance of dome-shaped structures and intracellular lipid droplets are characteristic of differentiated Caco-2 cells. When the Caco-2 cells are not dispersed equally during seeding, they will clump and overgrow in certain areas of the dish; and there will be a few areas in the dish without any cells. Swirling and placing the dish on a slanted surface should be avoided. It is also important to note that post-confluent Caco-2 cells are mo...

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.

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

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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.

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

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.

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

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

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

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

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

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

Using the optokinetic response to study visual function of zebrafish

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

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

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

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

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

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

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

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

Preparation of Segmented Microtubules to Study Motions Driven by the Disassembling Microtubule Ends

Microtubule depolymerization can provide force to transport different protein complexes and protein-coated beads in vitro. The underlying mechanisms are thought to play a vital role in the microtubule-dependent chromosome motions during cell division, but the relevant proteins and their exact roles are ill-defined. Thus, there is a growing need to develop assays with which to study such motility in vitro using purified components and defined biochemical milieu. Microtubules, however, are inherently unstable polymers; their switching between growth and shortening is stochastic and difficult to control. The protocols we describe here take advantage of the segmented microtubules that are made with the photoablatable stabilizing caps. Depolymerization of such segmented microtubules can be triggered with high temporal and spatial resolution, thereby assisting studies of motility at the disassembling microtubule ends. This technique can be used to carry out a quantitative analysis of the number of molecules in the fluorescently-labeled protein complexes, which move processively with dynamic microtubule ends. To optimize a signal-to-noise ratio in this and other quantitative fluorescent assays, coverslips should be treated to reduce nonspecific absorption of soluble fluorescently-labeled proteins. Detailed protocols are provided to take into account the unevenness of fluorescent illumination, and determine the intensity of a single fluorophore using equidistant Gaussian fit. Finally, we describe the use of segmented microtubules to study microtubule-dependent motions of the protein-coated microbeads, providing insights into the ability of different motor and nonmotor proteins to couple microtubule depolymerization to processive cargo motion.

Microtubules are inherently unstable polymers, and their switching between growth and shortening is stochastic and difficult to control. Here we describe protocols using segmented microtubules with photoablatable stabilizing caps. Depolymerization of segmented microtubules can be triggered with high temporal and spatial resolution, thereby assisting analysis of motions with the disassembling microtubule ends.

Microtubule depolymerization can provide force to transport different protein complexes and protein-coated beads in vitro. The underlying mechanisms are ...

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.

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.

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.

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.

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