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 ethylenediaminetetraacetic acid (EDTA) at 37 &#176;C until they are detached (15 min). To avoid cell clumping, mix the cells several times by gentle pipetting. Add 0.5 ml&#160;of the trypsinized cells to a 10 cm tissue culture dish containing 10 ml&#160;of pre-warmed growth media.</li>
		</ol>
		</li>
		<li>Gently shake the dishes several times in a forward and backward direction followed by a leftward and rightward direction. Avoid swirling the dishes as it may result in an unequal cell dispersion.</li>
		<li>Place the dishes on a flat surface in a 37 &#176;C incubator supplied with 5% CO2. Slanted surfaces may also result in an unequal cell dispersion.</li>
		<li>Change the growth media a day after starting the incubation.</li>
		<li>Monitor the cells and record the day they reach confluence. It will take about 1 week for the cells to reach confluence from the day they are split.</li>
		<li>Change the growth media twice a week before the cells reach confluence. Once the cells have reached confluence, change the growth media every other day. After the cells are 7-days post-confluent, change the growth media daily. 
		Note: The cells are ready for experiments when they are 13-days post-confluent (Figure 1). Since the cells will eventually become less effective in producing lipoproteins, use cells that are between 13 to 17-days post-confluent 8. Go to step 3 on how to conduct the experiment.</li>
	</ol>
	</li>
	<li>Using the permeable membrane system
	<ol>
		<li>Seed the Caco-2 cells in the permeable membrane insert as described in step 1.1.1.1. above. Avoid using cells from a frozen vial because their cell growth is normally slower (recovery period). Briefly, add 0.5 ml&#160;of the trypsinized cells to the apical chamber (upper compartment) that contains 10 ml&#160;of pre-warmed growth media. Also, add 10 ml&#160;of pre-warmed growth media to the basolateral chamber (lower compartment). 
		Note: For best practice, add the growth media to the apical chamber first and then to the basolateral chamber. When removing the old media, remove the basolateral media before the apical media. Avoid poking the polycarbonate membrane as it may rupture the membrane.</li>
		<li>Due to poor cell visibility through the polycarbonate membrane, seed the Caco-2 cells in a regular tissue culture dish with similar density, and use this dish to judge the cell confluence.</li>
		<li>Follow steps 1.1.2-1.1.6 above.</li>
	</ol>
	</li>
</ol>
2. Gene Overexpression
<ol>
	<li>Using the regular transfection approach
	<ol>
		<li>Seed the Caco-2 cells on a tissue culture dish as described in 1.1.1.1.-1.1.4 above. 
		Note: When the cells are about 40-50% confluent, they are ready to be transfected.</li>
		<li>Add 67 &#956;l&#160;of the transfection reagent to 472 &#956;l&#160;of the reduced serum media in a microcentrifuge tube. Avoid contact of the undiluted transfection reagent with the tube wall.</li>
		<li>Add 23 &#956;g of pLL3.7 enhanced green fluorescent protein (eGFP) 9 into the transfection reagent/reduced serum media mixture.</li>
		<li>Incubate the mixture for 20 min&#160;at RT.</li>
		<li>Replace the Caco-2 cells&#8217; growth media with 8 ml&#160;of 10% FBS in DMEM.</li>
		<li>Add the DNA/transfection reagent/reduced serum media mixture drop-wise to the dish.</li>
		<li>Gently shake the dishes several times in a forward and backward direction followed by a leftward and rightward direction.</li>
		<li>Place the dish in the 37 &#176;C incubator.</li>
		<li>Replace the media in the dish with 10 ml of growth media a day after starting the incubation.</li>
		<li>Monitor the gene expression.
		<ol>
			<li>Monitor the gene expression without 4&#8217;,6-diamidino-2-phenylindole (DAPI) staining. Using a fluorescent microscope, determine the percent of cells that are green. The percentage represents the transfection efficiency.</li>
			<li>Monitor the gene expression with DAPI staining.&#160;
			<ol>
				<li>Wash the cells with phosphate buffered saline (PBS) three times.</li>
				<li>Fix the cells by adding 10 ml&#160;of 4% formaldehyde in PBS (10 min incubation at RT).</li>
				<li>Wash the cells with PBS three times.</li>
				<li>Incubate the cells in the dark for 15 min&#160;at RT with 8 ml&#160;of PBS containing 1:500 DAPI, 1% BSA, 0.01% digitonin.</li>
				<li>Wash the cells with PBS three times.</li>
				<li>Using a fluorescent microscope, determine the number of cells that are green/blue relative to those that are blue (Figure 2 top panel). The calculated percentage represents the transfection efficiency.</li>
			</ol>
			</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Using the lentivirus overexpression system 10
	<ol>
		<li>Seed human embryonic kidney (HEK)293T cells in a 15 cm tissue culture dish (10% FBS in DMEM as their growth media). 
		Note: When the cells are about 60-70% confluent, they are ready to be transfected.</li>
		<li>Add 162 &#956;l&#160;of the transfection reagent to 1,133 &#956;l&#160;of the reduced serum media in a microcentrifuge tube 11. Avoid contact of the undiluted transfection reagent with the tube wall.</li>
		<li>Add 24 &#956;g of pLL3.7 eGFP (or another construct), 15.6 &#956;g of Rev response element (RRE), 6.0 &#956;g of REV, and 8.4 &#956;g of vesicular stomatitis virus glycoprotein (VSVG) into the transfection reagent/reduced serum media mixture.</li>
		<li>Incubate the mixture for 20 min&#160;at RT.</li>
		<li>Replace the growth media with 16 ml&#160;of 10% FBS in DMEM.</li>
		<li>Add the DNA/transfection reagent/reduced serum media mixture drop-wise to the dish.</li>
		<li>Gently shake the dishes several times in a forward and backward direction followed by a leftward and rightward direction. It is normal to see some of the HEK293T cells detached at this point.</li>
		<li>Place the dish in the incubator.</li>
		<li>After 24 hr&#160;of incubation, replace the media in the dish with 25 ml&#160;of growth media.</li>
		<li>On the following day, collect the growth media containing the lentivirus (first collection).</li>
		<li>Repeat step 2.2.10 to collect more lentivirus (the second collection).</li>
		<li>Pool the first and the second collection, and remove cell debris by centrifugation (2,500 x g for 5 min).</li>
		<li>Filter the collection with a disposable bottle-top filter (0.45 &#956;m pore), and concentrate the virus by centrifuging the collection at 31,400 x g for 2 hr&#160;at RT (JA-20 rotor). Mark the bottom of the centrifuge tube where the pellet is located. Decant the supernatant, and leave the tube inverted at RT for about 15 min.</li>
		<li>Resuspend the virus-containing pellet with 100 &#956;l&#160;of 1X PBS. Avoid bubbles.</li>
		<li>Store the concentrated virus at -80 &#176;C. Avoid freeze-thaw cycles.</li>
		<li>Determine the optimal amount of virus needed to transduce the Caco-2 cells by titration. To save cost, use a 24-well plate (0.3 ml&#160;of growth media/well) instead of a 10 cm tissue culture dish for titration.
		<ol>
			<li>Add increasing amounts of the concentrated virus (0, 1, 2, 5, 10, 25, and 50 &#956;l/well) supplemented with polybrene (final concentration = 5 &#956;g/ml) to the 40-70% confluent Caco-2 cells. Shake the 24-well plate gently as described in 1.1.2.</li>
			<li>Monitor their gene expression (Step 2.1.10.) (Figure 2 bottom 4 panels).</li>
		</ol>
		</li>
		<li>Since the expression of transgene is generally sustained, maintain the transduced cells for future experiments and continuously monitor their gene expression (Step 2.1.10.).</li>
	</ol>
	</li>
</ol>
3. Stimulating the Lipoprotein Secretion
<ol>
	<li>Using the regular tissue culture dish
	<ol>
		<li>Prepare a 100X lipid mixture by mixing 50 mg of oleic acid, 40 mg of lecithin, and 48 mg of sodium taurocholate. Bring the volume to 900 &#956;l&#160;with PBS (enough for 8 dishes). Vortex the lipid mixture vigorously.</li>
		<li>Add 900 &#956;l&#160;of the 100X lipid mixture into 89.1 ml&#160;of growth media. Mix, and filter the lipid-containing media. The final concentrations (1X) of oleic acid, lecithin, and sodium taurocholate are 2.0 mM, 1.36 mM, and 1.0 mM, respectively.</li>
		<li>Add 10 ml&#160;of the lipid-containing media to the cells.</li>
		<li>Incubate the cells with the lipid-containing media for 4 hr&#160;in a 37 &#176;C incubator.</li>
		<li>Wash the cells with PBS three times.</li>
		<li>Add 10 ml&#160;of the growth media to the cells to collect the lipoprotein secretion.</li>
		<li>Incubate for 2 hr&#160;in a 37 &#176;C incubator.</li>
		<li>Collect the lipoprotein-containing media.</li>
	</ol>
	</li>
	<li>Using the permeable membrane system
	<ol>
		<li>Follow steps 3.1.1-3.1.2 to prepare the lipid mixture.</li>
		<li>Add 10 ml&#160;of the lipid-containing media to the apical chamber and 10 ml&#160;of the growth media to the basolateral chamber.</li>
		<li>Incubate for 4 hr&#160;in a 37 &#176;C incubator. Collect the lipoprotein-containing media in the basolateral chamber.</li>
	</ol>
	</li>
</ol>
4. Lipoprotein Isolation (Figure 3)
<ol>
	<li>Remove the cell debris from the lipoprotein-containing media by centrifugation (2,000 x g for 5 min).</li>
	<li>Decant the media into a 50 ml&#160;tube.</li>
	<li>Add 5.95 g of sodium chloride to the media.</li>
	<li>If the sample will be analyzed by the TEM, add 1 protease inhibitor cocktail tablet to maintain the integrity of the lipoproteins.</li>
	<li>Bring the volume to 23 ml&#160;with water (1.2 g/ml&#160;NaCl density solution).</li>
	<li>Dissolve the solutes completely.</li>
	<li>Decant all of the solution into a polycarbonate ultracentrifuge tube.</li>
	<li>Gently overlay the 1.2 g/ml&#160;density solution with 0.5 ml&#160;water (1.0 g/ml&#160;density).</li>
	<li>Balance the tube by weight and not by volume.</li>
	<li>Gently load the tubes in the T865 rotor.</li>
	<li>Spin the samples in the ultracentrifuge at 429,460 x g for 24 hr&#160;at 4 &#176;C.</li>
	<li>Immediately isolate the top 0.5 ml&#160;solution by gentle pipetting. Keep the tube as still as possible.</li>
</ol>
5. TEM Analysis
<ol>
	<li>Prepare 5 ml&#160;of 2% phosphotungstic acid (w/v) and adjust the pH to 6.0.</li>
	<li>Filter the 2% phosphotungstic acid with a syringe filter (pore size: 0.2 &#181;m).</li>
	<li>Drop 20 &#956;l&#160;of the lipoprotein sample on a sterile dish.</li>
	<li>Drop 20 &#956;l&#160;of the filtered 2% phosphotungstic acid next to the sample on the same dish.</li>
	<li>Gently drop an EM grid with the dull side resting on the sample.</li>
	<li>Incubate at RT for 1 min.</li>
	<li>Gently tap the side of the EM grid on a filter paper to remove the sample from the grid.</li>
	<li>Gently drop the EM grid with the dull side resting on the 2% phosphotungstic acid.</li>
	<li>Incubate at RT for 1 min.</li>
	<li>Gently tap the side of the EM grid on a filter paper to remove the phosphotungstic acid from the grid.</li>
	<li>Using a TEM, capture the images of the lipoproteins (Figure 4).</li>
</ol>

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

The author has nothing to disclose.

In 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...

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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 ...

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

Research

JoVE Journal

Biology

18.6K Views.  California Northstate University. Caco-2 cells can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs/vitamins. The permeable membrane system separates the apical from the basolateral compartment, while the lentivirus expression system offers an effective gene overexpression method. The isolation of lipoproteins is confirmed by TEM.

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

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

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

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

Badania wizualne Wrażliwość na prędkość i kierunek ruchu w Jaszczurki

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 ...

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 ...

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) ...

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

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

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

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

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

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

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

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

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

Investigating Zinc Transport Mechanism in Mammalian Cells with FluoZin-3 Assay

Characterizing Mammalian Zinc Transporters Using an In Vitro Zinc Transport Assay

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

Author Spotlight: Exploring Cellular Zinc Regulation Through ZnT1 Functionality 

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

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