This work was supported by a Scientist Development Grant (11SDG5190025) from the American Heart Association (to A.R.-D.), and by National Institute of Health R01 grants AA-023764 (to A.N.B.), and HL-104631 and R37 AA-11560 (to A.M.D).

All experimental procedures with animals were performed at the University of Tennessee Health Science Center (UTHSC). The care of animals and experimental protocols were reviewed and approved by the Animal Care and Use Committee of the UTHSC, which is an institution accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International.
1. Enrichment of tissues and cells using methyl-&#946;-cyclodextrin saturated with cholesterol
NOTE: The cholesterol enrichment protocol below is suitable for tissues, cells, and cell lines. As an example, we describe the steps performed for enriching mammalian cerebral arteries. Representative results are provided for both cerebral arteries (Figure 1) and neurons (Figure 4).
<ol>
	<li>Preparation of M&#946;CD saturated with cholesterol
	<ol>
		<li>Weigh 0.064 g of M&#946;CD and dissolve it in a flask containing 10 mL of phosphate-buffered saline (PBS) solution to obtain a final concentration of 5 mM M&#946;CD. Stir the solution with a stir bar to ensure that the M&#946;CD is fully dissolved.</li>
		<li>Weigh 0.0024 g of cholesterol powder and add it to the&#160;same flask to obtain a 0.63 mM cholesterol concentration. Then stir the solution vigorously. Use a spatula to break up as many cholesterol chunks as possible (some chunks will remain until incubation).</li>
		<li>Cover the flask with at least two layers of paraffin film and shake slowly (~30 oscillations/min) in a 37 &#176;C water bath overnight. This step is critical.</li>
		<li>After 8-16 h, cool the solution to room temperature (RT), and then filter it through a 0.22 &#956;m polyethersulfone syringe filter into a glass bottle. 
		NOTE: To reach different cholesterol concentrations in solution, adjust the amounts of both cholesterol and M&#946;CD by simple proportion. It is important to maintain the M&#946;CD:cholesterol molar ratio at 8:1 to obtain saturation of methyl-&#946;-cyclodextrin carrier with cholesterol. The cholesterol-enriching solution can be used immediately or over the course of several days if stored at 4 &#176;C. However, the cholesterol-enriching ability declines over time as the cholesterol aggregates appear, and the solution becomes cloudy.</li>
	</ol>
	</li>
	<li>Treatment of cerebral arteries with M&#946;CD saturated with cholesterol
	<ol>
		<li>Euthanize a Sprague Dawley rat (250-300 g) by placing it in a chamber with 2% isoflurane. Then, decapitate the anesthetized rat using a sharp guillotine or a large sharp pair of scissors. 
		NOTE: If performing these procedures regularly, it is useful to develop a schedule for guillotine sharpening. Also, a separate pair of scissors should be dedicated for rodent decapitation. Rodent decapitation is a terminal procedure; therefore, the instruments do not have to be sterile. Cleaning with soapy water after each use is sufficient.</li>
		<li>Position the rat&#39;s head facing forward, away from the researcher. Place the pointed part of a medium sized pair of scissors between the skull and the brain stem, and cut laterally on both sides.</li>
		<li>Use forceps to pry the top skull open by pulling up on the base of the skull where the lateral cuts were made and carefully remove the brain. Make sure to cut the optical nerves that hold the brain within the skull.</li>
		<li>Put the brain in a beaker with PBS on ice after removal. 
		NOTE: The brain can be stored on ice for 4-6 h at 4 &#176;C.</li>
		<li>In a nonsterile environment transfer the rat brain to a waxed dissection bowl with enough PBS to submerge it. Pin the brain down to keep it from moving. 
		NOTE: Step 1.2.5 can be carried out at RT if performed quickly. Otherwise, it needs to be done on ice.</li>
		<li>Use sharp forceps and small surgical scissors to dissect the cerebral arteries and their branches that form the Circle of Willis at the base of the brain under the microscope in PBS at RT. Be gentle when dissecting to ensure that the artery tissue is not stretched or cut. This step is critical.</li>
		<li>Briefly rinse the artery segments (up to 1 cm long) in PBS either in a 96 well plate or in a 35 mm dish to remove the leftover blood, and then place them for 10 min into enough of the cholesterol-enriching solution (prepared in step 1.1) to cover the entire artery segments. Use a 35 mm dish if there is an ample amount of cholesterol-enriching solution and a 96 well plate if the arteries are small or if there is a shortage of cholesterol-enriching solution. 
		NOTE: The same approach can be used to enrich other tissues and cells with cholesterol using a 60 min incubation time. For example, this approach has been previously used for cholesterol enrichment of mouse cerebral arteries35,45, hippocampal neurons32, atrial myocytes37, and HEK 293 cells39. The minimal incubation time needs to be determined for each tissue or cell type based on the validation of cholesterol enrichment at different time points with a cholesterol-sensitive assay (e.g., the biochemical determination of the amount of cholesterol in the tissue by staining with the cholesterol-sensitive fluorescence dye filipin).</li>
	</ol>
	</li>
	<li>Stain the artery tissue with the steroid-sensitive fluorescence dye filipin to determine any alterations in cholesterol levels. 
	NOTE: In the Representative Results section, we demonstrate the results of two approaches to assess changes in cholesterol levels: a biochemical assay performed through the application of a commercially available cholesterol oxidase-based kit (see Table of Materials) and staining with the steroid-sensitive fluorescence dye filipin. The first approach can be performed by following the manufacturer&#39;s instructions. The protocol for the latter approach is provided below.
	<ol>
		<li>Using a fresh bottle of filipin powder, prepare a 10 mg/mL stock solution in dimethyl sulfoxide (DMSO). This step is critical. 
		NOTE: The resulting solution is light-sensitive. If prepared correctly, the filipin stock solution is yellowish. Some filipin powder may stick to the bottle cap. Therefore, it is important to rinse the bottle and cap with DMSO solvent to retain the entire amount of filipin. Once prepared, filipin stock must be used within several days. Filipin completely loses its fluorescence ability after 5 days, even when stored in the dark at -20 &#176;C.</li>
		<li>Remove the artery segments from the cholesterol-enriching solution and wash them 3x with PBS for 5 min.</li>
		<li>Fix the artery segments in 4% paraformaldehyde for 15 min on ice. 
		CAUTION: Paraformaldehyde is light-sensitive. Therefore, work must be carried out in the dark.</li>
		<li>Place the artery segments into 0.5% Triton in PBS at RT for 10 min to permeabilize the tissue and facilitate dye penetration.</li>
		<li>Wash the artery segments 3x with PBS for 5 min on a shaker. This step is critical. 
		NOTE: When the Triton has been completely washed out, there should not be any bubbles on the surface of the PBS solution.</li>
		<li>Dilute the filipin stock solution in PBS to a final concentration of 25 &#956;g/mL. Remove the arteries form the PBS solution and place them in the diluted filipin solution for 1 h in the dark. This step is critical.</li>
		<li>Wash out the filipin by rinsing the artery segments 3x with PBS for 5 min on a shaker. This step is critical.</li>
		<li>Rinse the artery segments briefly with distilled water, absorb excessive liquid with a paper napkin, and mount the arteries on a slide using commercially available mounting media (see Table of Materials).</li>
		<li>Cover the artery with a coverslip avoiding rolling or twisting of the artery and set the slides to dry in a dark area at RT for 24 h.</li>
		<li>After the mounting media dries, seal the coverslip edges with clear nail polish, and leave the nail polish to dry for 10-15 min. Store the slides in the dark at -20 &#176;C.</li>
		<li>Equilibrate the slides to RT before imaging.</li>
		<li>Image the tissue with a fluorescence microscope or a fluorescence reader with the excitation set at 340-380 nm and emission at 385-470 nm. 
		CAUTION: Filipin photobleaches quickly; thus, samples have to be imaged promptly.</li>
	</ol>
	</li>
</ol>
2. Enrichment of Xenopus oocytes using cholesterol-enriched phospholipid-based dispersions (liposomes)
<ol>
	<li>Preparation of solutions
	<ol>
		<li>To prepare a stock solution of cholesterol, dissolve 10 mg of cholesterol powder in 1 mL of chloroform in a 10 mL glass beaker or bottle. Transfer the solution into a 1.5 mL capped glass bottle. 
		CAUTION: In view of the toxicity and rapid evaporation of chloroform, work in the hood and keep reagents on ice.</li>
		<li>Prepare a 150 mM KCl, 10 mM Tris-HEPES, pH = 7.4 buffer for cholesterol-enriched phospholipids. To do so, dissolve in an Erlenmeyer flask 5.5905 g of KCl and 0.6057 g of Tris in double-distilled water to a total of 0.5 L volume. In another flask, dissolve 5.5905 g of KCl and 1.19155 g of HEPES in double-distilled water to a total of 0.5 L volume. Mix the two solutions together in a 1 L Erlenmeyer flask, and adjust the pH to 7.4 with HCl. 
		NOTE: Store the resulting 150 mM KCl, 10 mM Tris-HEPES solution at 4 &#176;C.</li>
		<li>To prepare ND96 pre-medium oocyte culturing (low K+, low Ca2+) buffer, combine 1 mL of 2 M KCl, 1 mL of 1 M MgCl2, 45.5 mL of 2 M NaCl, and 5 mL of 1/1 M NaOH-HEPES in a 1 L Erlenmeyer flask. Add 900 mL double-distilled water and adjust the pH to 7.4 with HCl. Transfer the solution to a 1 L cylinder and bring the volume to 1 L with double-distilled water. Then add 1.8 mL of 1 M CaCl2 and filter the solution. 
		NOTE: Slight variations in the ratios between the components used to make an ND96 solution do not seem to be critical for cholesterol enrichment, possibly because the ND96 solution is not used during the enrichment step itself but for storage. An example is a 1 L solution that has a slightly lower concentration of sodium and chloride ions, and is made by combining 2 mL of 1 M KCl, 1 mL of 1 M MgCl2, 82.5 mL of 1 M NaCl, 5 mL of 1 M HEPES, and 1.8 mL of 1 M CaCl2 (Ca2+ is omitted to obtain a Ca2+ free solution). Adjust the pH of the solution to 7.4 with NaOH. Store the resulting ND96 oocyte culturing solution at 4 &#176;C for up to 1 month.</li>
	</ol>
	</li>
	<li>Preparation of the phospholipid-based dispersion with cholesterol liposomes
	<ol>
		<li>In a 12 mL glass tube, combine 200 &#956;L of each of the following 10 mg/mL chloroform-dissolved lipid solutions: L-&#945;-phosphatidylethanolamine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine, and cholesterol.</li>
		<li>Evaporate the chloroform in the hood to dry slowly under a stream of nitrogen. This step is critical.</li>
		<li>Suspend the lipids in 800 &#956;L of buffered solution consisting of 150 mM KCl and 10 mM Tris-HEPES at pH = 7.4, and cover with paraffin film.</li>
		<li>Sonicate gently at 80 kHz for 10 min until a milky mixture is formed. This step is critical. 
		CAUTION: When sonicating, the dispersion in the glass tube should vibrate gently, forming small waves. Drops of dispersion should not be jumping within the tube.</li>
	</ol>
	</li>
	<li>Enriching Xenopus oocytes with cholesterol 
	NOTE: Frog oocyte-containing ovaries can be obtained from two sources: First, Xenopus laevis female frogs can be housed for the purpose of in-house surgery. This procedure must be approved by the Institutional Animal Care and Use Committee. Second, whole ovaries can be purchased from commercial suppliers. As an alternative to purchasing or isolating whole ovaries and then digesting them as described in steps 2.3.1-2.3.4, individual oocytes are available commercially for purchase. If the latter are used, steps 2.3.1-2.3.4 can be skipped.
	<ol>
		<li>Keep the freshly obtained ovaries at ~14 &#176;C in an ND96 solution. Under these conditions, the ovaries can be stored for up to 1 week.</li>
		<li>To obtain individual oocytes, disrupt the ovarian sac in multiple places using sharp forceps. Place the ovary chunks into a 60 mm plate, with 5 mL of Ca2+-free ND96 supplemented with 0.5 mg/mL collagenase. Shake on an orbital shaker at 60 oscillations/min for 15 min at RT. 
		NOTE: This step will ensure the digestion of the ovarian sac. To preserve enzymatic activity, avoid storing collagenase containing ND96 for extended periods of time (&#62;1 h). Even brief storage should be performed at cool temperatures of under 15 &#176;C.</li>
		<li>Using a transfer pipette with a wide tip, vigorously pipette the oocyte-containing solution up and down approximately 5-10x to separate individual oocytes. At this step, the solution will turn dark.</li>
		<li>Quickly rinse the oocytes with Ca2+-free ND96 until the solution becomes transparent.</li>
		<li>Transfer individual oocytes to Ca2+-containing ND-96 solution supplemented with 2 mg/mL of gentamicin using a transfer pipette with a narrow tip. 
		NOTE: This step is essential when it is necessary to store the oocytes. Individual oocytes can be stored in an incubator for several days at 14-17 &#176;C. However, dead oocytes that are whitish must be removed at least once a day to avoid contamination of the solution with toxic chemicals.</li>
		<li>Transfer 90 &#956;L of the cholesterol-enriched phospholipid-based dispersion into one well of a 96 well plate.</li>
		<li>Transfer up to six oocytes from the ND96 medium to the well with as little medium as possible. This step is critical. 
		CAUTION: Do not expose the oocytes to the air during the transfer to keep the oocytes intact.</li>
		<li>Place the 96 well plate on a three-dimensional platform rotator to provide a small orbital motion to the oocytes in the cell for 5-10 min.</li>
		<li>Add a drop of ND96 into the well, and transfer the cholesterol-enriched oocytes from the 96 well plate to a 35 mm plate with ND96 for immediate use. This step is critical.</li>
		<li>Use a commercially available cholesterol oxidase-based kit (see Table of Materials) to assess changes in cholesterol levels by following the manufacturer&#39;s instructions.</li>
	</ol>
	</li>
</ol>

Dr. A. M. Dopico is a special, part time, federal employee and current member of The National Advisory Council on Alcohol Abuse and Alcoholism.

Methods to enrich mammalian tissues and cells and Xenopus oocytes with cholesterol constitute a powerful tool for investigating the effect of elevated cholesterol levels on individual molecular species, on complex macromolecular systems (e.g., proteins), and on cellular and organ function. In this paper, we have described two complementary approaches that facilitate such studies. First, we described how to enrich tissues and cells with cholesterol using M&#946;CD saturated with cholesterol. We demonstrated that ...

Cholesterol, a major cellular lipid, plays numerous critical functional and structural roles1,2,3,4,5,6,7,8,9. From regulating the physical properties of the plasma membrane to ensuring cell viability, growth, proliferation, and serving as a signaling and precursor molecule in a plethora of biochemical pathways, cholesterol is an imperative component necessary for normal cell and organ function. As a result, cholesterol deficiency results in severe physical malformations and a variety of disorders. On the other hand, even a small increase in cholesterol above physiological levels (2-3x) is cytotoxic1,2,10 and has been associated with the development of disorders, including cardiovascular11,12,13 and neurodegenerative diseases14,15,16,17. Thus, to interrogate the critical functions of cholesterol and to determine the effect of changes in cholesterol levels, different approaches that alter the content of cholesterol in tissues, cells, and Xenopus oocytes have been developed.
Alteration of cholesterol levels in mammalian tissues and cells 
Several approaches can be harnessed to decrease the levels of cholesterol in tissues and cells18. One approach involves their exposure to statins dissolved in lipoprotein-deficient serum to inhibit HMG-CoA reductase, which controls the rate of cholesterol synthesis19,20. However, these cholesterol lowering drugs also inhibit the formation of non-sterol products along the mevalonate pathway. Therefore, a small amount of mevalonate is added to allow the formation of these products21 and enhance the specificity of this approach. Another approach for decreasing cholesterol levels involves the use of &#946;-cyclodextrins. These glucopyranose monomers possess an internal hydrophobic cavity with a diameter that matches the size of sterols22, which facilitates the extraction of cholesterol from cells, thereby depleting them from their native cholesterol content23. An example is 2-hydroxypropyl-&#946;-cyclodextrin (HP&#946;CD), a preclinical drug currently being tested for treatment of the Niemann-Pick type C disease, a genetically inherited fatal metabolic disorder characterized by lysosomal cholesterol storage24. The level of cholesterol depletion depends on the specific derivative used. For example, HP&#946;CD extracts cholesterol with a lower capacity than the methylated derivative, methyl-&#946;-cyclodextrin (M&#946;CD)24,25,26,27,28,29,30. Notably, however, &#946;-cyclodextrins can also extract other hydrophobic molecules in addition to cholesterol, which may then result in nonspecific effects31. In contrast to depletion, cells and tissues can be specifically enriched with cholesterol through treatment with &#946;-cyclodextrin that has been presaturated with cholesterol23. This approach can also be used as a control for the specificity of &#946;-cyclodextrins used for cholesterol depletion31. Depletion of cholesterol from tissues and cells is straightforward and can be achieved by exposing the cells for 30-60 min to 5 mM M&#946;CD dissolved in the medium used for storing the cells. This approach can result in a 50% decrease in cholesterol content (e.g., in hippocampal neurons32, rat cerebral arteries33). On the other hand, preparing the &#946;-cyclodextrin-cholesterol complex for cholesterol enrichment of tissue and cells is more complex, and will be described in the protocol section.
An alternative approach to enriching tissues and cells using &#946;-cyclodextrin saturated with cholesterol involves the use of LDL, which relies on LDL receptors expressed in the tissues/cells18. While this approach offers the advantage of using the natural cholesterol homeostasis machinery of the cell, it has several limitations. First, tissues and cells that do not express the LDL receptor cannot be enriched using this approach. Second, LDL particles contain other lipids in addition to cholesterol. Specifically, LDL is comprised of the protein ApoB100 (25%) and the following lipids (75%): ~6-8% cholesterol, ~45-50% cholesteryl ester, ~18-24% phospholipids, and ~4-8% triacylglycerols34. Thus, delivery of cholesterol via LDL particles is nonspecific. Third, the percentage of increase in cholesterol content by LDL in tissues and cells that express the LDL receptor may be significantly lower than the increase observed using cyclodextrin saturated with cholesterol. For example, in a previous study, enrichment of rodent cerebral arteries with cholesterol via LDL resulted in only a 10-15% increase in cholesterol levels35. In contrast, enrichment of these arteries with cyclodextrin saturated with cholesterol as described in the protocol section resulted in &#62;50% increase in the cholesterol content (See Representative Results section, Figure 1).
Alteration of cholesterol levels in Xenopus oocytes 
Xenopus oocytes constitute a heterologous expression system commonly used for studying cell and protein function. Earlier studies have shown that the cholesterol to phospholipid molar ratio in Xenopus oocytes is 0.5 &#177; 0.136. Due to this intrinsic high level of cholesterol, increasing the content of cholesterol in this system is challenging, yet can be achieved using dispersions made from membrane phospholipids and cholesterol. The phospholipids that we have chosen for this purpose are similar to those used for forming artificial planar lipid bilayers and include L-&#945;-phosphatidylethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (POPS), as described in the protocol section. This approach can result in &#62;50% increase in cholesterol content (See Representative Results section, Figure 2).
An alternative approach to enriching Xenopus oocytes with phospholipid-based dispersions involves the use of cyclodextrin saturated with cholesterol, which is similar to the way tissues and cells are enriched. However, we have found this approach to be of low reproducibility and efficiency, with an average of ~25% increase in cholesterol content. This is possibly due to the different loading capacity of these two approaches (See Representative Results section, Figure 3). In contrast, it has been shown that using cyclodextrin to deplete cholesterol from Xenopus oocytes can result in a ~40% decrease in cholesterol content36.
Here, we focus on cholesterol enrichment of mammalian tissues and cells through the application of cyclodextrin saturated with cholesterol, and of Xenopus oocytes using liposomes. Both approaches can be harnessed to delineate the effect of increased levels of cholesterol on protein function. The mechanisms of cholesterol modulation of protein function may involve direct interactions8 and/or indirect effects9. When cholesterol affects protein function via direct interactions, the effect of an increase in cholesterol levels on protein activity is likely independent of the cell type, expression system, or enrichment approach. For example, we utilized these two approaches to determine the effect of cholesterol on G-protein gated inwardly rectifying potassium (GIRK) channels expressed in atrial myocytes37, hippocampal neurons32,38, HEK29339 cells, and Xenopus oocytes32,37. The results obtained in these studies were consistent: in all three types of mammalian cells and in amphibian oocytes cholesterol upregulated GIRK channel function (see Representative Results section, Figure 4, for hippocampal neurons and the corresponding experiments in Xenopus oocytes). Furthermore, the observations made in these studies were also consistent with the results of studies carried out in atrial myocytes37,40 and hippocampal neurons32,38 freshly isolated from animals subjected to a high cholesterol diet40. Notably, cholesterol enrichment of hippocampal neurons using M&#946;CD reversed the effect of atorvastatin therapy used for addressing the impact of the high cholesterol diet both on cholesterol levels and GIRK function38. In other studies, we investigated the effect of mutations on cholesterol sensitivity of the inwardly rectifying potassium channel Kir2.1 using both Xenopus oocytes and HEK293 cells41. Again, the effect of the mutations on the sensitivity of the channel was similar in the two systems.
The applications of both enrichment methods for determining the impact of elevated cholesterol levels on molecular, cellular, and organ function are numerous. In particular, the use of cyclodextrin-cholesterol complexes to enrich cells and tissues is very common largely due to its specificity. Recent examples of this approach include the determination of the impact of cholesterol on HERG channel activation and underlying mechanisms42, the discovery that cholesterol activates the G protein coupled receptor Smoothened to promote Hedgehog signaling43, and the identification of the role of cholesterol in stem cell biomechanics and adipogenesis through membrane-associated linker proteins44. In our own work, we utilized mammalian tissue enrichment with the M&#946;CD:cholesterol complex to study the effect of cholesterol enrichment on basic function and the pharmacological profile of calcium- and voltage-gated channels of large conductance (BK, MaxiK) in vascular smooth muscle35,45,46. In other studies, we used the phospholipid-based dispersion approach for enriching Xenopus oocytes with cholesterol to determine the roles of different regions in Kir2.1 and GIRK channels in cholesterol sensitivity41,47,48,49, as well as to determine putative cholesterol binding sites in these channels32,50,51.

<table><tbody><tr><td>Amplex Red Cholesterol Assay Kit</td><td>Invitrogen</td><td>A12216</td><td></td></tr><tr><td>Pierce BCA Protein Assay Kit</td><td>Thermo Scientific</td><td>23225</td><td></td></tr><tr><td>Pre-Diluted Protein Assay Standards BSA set</td><td>Thermo Scientific</td><td>23208</td><td></td></tr><tr><td>Brain PE 25Mg in Chloroform</td><td>Avanti Lipids</td><td>840022C</td><td></td></tr><tr><td>16:0-18:1 PS 25Mg Chloroform</td><td>Avanti Lipids</td><td>840034C</td><td></td></tr><tr><td>Cholesterol 100Mg Powder</td><td>Sigma</td><td>C8667</td><td></td></tr><tr><td>KCl</td><td>Fisher</td><td>P217</td><td></td></tr><tr><td>Trizma base</td><td>Sigma</td><td>T6066</td><td></td></tr><tr><td>HEPES</td><td>Corning</td><td>61-034-RO</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Fisher</td><td>M33</td><td></td></tr><tr><td>NaCl</td><td>Fisher</td><td>S271</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher</td><td>P285</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>EMD Chemicals</td><td>MX0070-1</td><td></td></tr><tr><td>EDTA</td><td>VWR</td><td>E177</td><td></td></tr><tr><td>Dextrose Anhydrous</td><td>Fisher</td><td>BP350</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma</td><td>S6014</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>C3881</td><td></td></tr><tr><td>Blood Gas Tank</td><td>nexAir</td><td></td><td></td></tr><tr><td>NaOH</td><td>Fisher</td><td>S318</td><td></td></tr><tr><td>1.5mL tubes</td><td>Fisher</td><td>S35818</td><td></td></tr><tr><td>Gastight Syringe 100uL</td><td>Hamilton</td><td>1710</td><td></td></tr><tr><td>Microliter Syringe 25uL</td><td>Hamilton</td><td>702</td><td></td></tr><tr><td>12 mL heavy duty conical centrifuge beaded rim tube</td><td>Pyrex</td><td>8120-12</td><td></td></tr><tr><td>Chloroform</td><td>Fisher</td><td>C298</td><td></td></tr><tr><td>Support Stand</td><td>Homescience Tools</td><td>CE-STAN5X8</td><td></td></tr><tr><td>Universal Clamp, 3-Prong</td><td>Homescience Tools</td><td>CE-CLPUNIV</td><td></td></tr><tr><td>Sonicator</td><td>Laboratory Supplies</td><td>G112SP1G</td><td></td></tr><tr><td>3D rotator mixer</td><td>Benchmark Scientific</td><td>B3D 1308</td><td></td></tr><tr><td>96 well plate</td><td>Sigma</td><td>BR781602</td><td></td></tr><tr><td>N2 gas</td><td>nexAir</td><td></td><td></td></tr><tr><td>Glass beakers 40ml-1L</td><td>Fisher</td><td>02-540</td><td></td></tr><tr><td>Ice Machine</td><td>Scotsman</td><td>CU1526MA-1</td><td></td></tr><tr><td>Ice bucket</td><td>Fisher</td><td>50-136-7764</td><td></td></tr><tr><td>1X PBS</td><td>Corning</td><td>21-031-CM</td><td></td></tr><tr><td>TritonX</td><td>Fisher</td><td>BP151-100</td><td></td></tr><tr><td>Sonic Dismembrator</td><td>Fisher</td><td>Model 100</td><td></td></tr><tr><td>Eppendorf microcentrifuge</td><td>Eppendorf</td><td>Model 5417R</td><td></td></tr><tr><td>Amber bottles</td><td>Fisher</td><td>03-251-420</td><td></td></tr><tr><td>Corning&amp;trade; Disposable Glass Pasteur Pipets</td><td>FIsher</td><td>13-678-4A</td><td></td></tr><tr><td>Parafilm</td><td>FIsher</td><td>50-998-944</td><td></td></tr><tr><td>Isotemp&amp;trade; BOD Refrigerated Incubator</td><td>FIsher</td><td>97-990E</td><td></td></tr><tr><td>Oocytes</td><td>Xenoocyte&amp;trade;</td><td>10005</td><td></td></tr><tr><td>Rat</td><td>Envigo</td><td>Sprague Dawley</td><td>weight 250g</td></tr><tr><td>Methyl-&amp;beta;-cyclodextrin</td><td>Sigma</td><td>C4555</td><td></td></tr><tr><td>Water bath incubator with shaker</td><td>Precision</td><td>51221080</td><td>Lowest shaker setting O/N 37 &amp;deg;C</td></tr><tr><td>Filipin</td><td>Sigma</td><td>SAE0088-1ML</td><td></td></tr><tr><td>DMSO</td><td>Fisher</td><td>BP231</td><td></td></tr><tr><td>Paraformaldehyde 4%</td><td>Mallinckrodt</td><td>2621</td><td></td></tr><tr><td>DI H&lt;sub&gt;2&lt;/sub&gt;O</td><td>University DI source</td><td></td><td></td></tr><tr><td>ProLong Gold antifade reagnet</td><td>Invitrogen</td><td>P10144</td><td></td></tr><tr><td>Microslides 75x25mm Frosted</td><td>Diagger</td><td>G15978A</td><td></td></tr><tr><td>Forceps</td><td>Fine Science Tools</td><td>11255-20</td><td></td></tr><tr><td>Microscope Coverslip</td><td>Diagger</td><td>G15972B</td><td></td></tr><tr><td>Clear nail polish</td><td>Revlon</td><td>771 Clear</td><td></td></tr><tr><td>Labeling Tape</td><td>Fisher</td><td>15-901-20F</td><td></td></tr><tr><td>Securline Lab Marker II</td><td>Sigma</td><td>Z648205-5EA</td><td></td></tr><tr><td>BD 10mL Syringe</td><td>Fisher</td><td>14-823-16E</td><td></td></tr><tr><td>1.2 &amp;mu;m syringe filter</td><td>VWR</td><td>28150-958</td><td></td></tr><tr><td>KimWipes</td><td>Fisher</td><td>06-666A</td><td></td></tr><tr><td>pH probe</td><td>Sartorus</td><td>py-p112s</td><td></td></tr><tr><td>pH meter</td><td>Denver instrument</td><td>Model 225</td><td></td></tr><tr><td>70% ETOH</td><td>Pharmco</td><td>211USP/NF</td><td></td></tr><tr><td>Timer</td><td>Fisher</td><td>02-261-840</td><td></td></tr><tr><td>Steno book</td><td>Staples</td><td>163485</td><td></td></tr></tbody></table>

enrichment of mammalian tissues and xenopus oocytes with cholesterol

Cholesterol enrichment of mammalian tissues and cells, including Xenopus oocytes used for studying cell function, can be accomplished using a variety of methods. Here, we describe two important approaches used for this purpose. First, we describe how to enrich tissues and cells with cholesterol using cyclodextrin saturated with cholesterol using cerebral arteries (tissues) and hippocampal neurons (cells) as examples. This approach can be used for any type of tissue, cells, or cell lines. An alternative approach for cholesterol enrichment involves the use of low-density lipoprotein (LDL). The advantage of this approach is that it uses part of the natural cholesterol homeostasis machinery of the cell. However, whereas the cyclodextrin approach can be applied to enrich any cell type of interest with cholesterol, the LDL approach is limited to cells that express LDL receptors (e.g., liver cells, bone marrow-derived cells such as blood leukocytes and tissue macrophages), and the level of enrichment depends on the concentration and the mobility of the LDL receptor. Furthermore, LDL particles include other lipids, so cholesterol delivery is nonspecific. Second, we describe how to enrich Xenopus oocytes with cholesterol using a phospholipid-based dispersion (i.e., liposomes) that includes cholesterol. Xenopus oocytes constitute a popular heterologous expression system used for studying cell and protein function. For both the cyclodextrin-based cholesterol enrichment approach of mammalian tissue (cerebral arteries) and for the phospholipid-based cholesterol enrichment approach of Xenopus oocytes, we demonstrate that cholesterol levels reach a maximum following 5 min of incubation. This level of cholesterol remains constant during extended periods of incubation (e.g., 60 min). Together, these data provide the basis for optimized temporal conditions for cholesterol enrichment of tissues, cells, and Xenopus oocytes for functional studies aimed at interrogating the impact of cholesterol enrichment.

The use of cyclodextrin saturated with cholesterol as a means for enriching tissues and cells with cholesterol is well established. Here, we first demonstrate the application of this widely used approach for enriching rat cerebral arteries with cholesterol using M&#946;CD saturated with cholesterol. Figure 1A shows an example of an imaged cerebral artery smooth muscle layer and demonstrates the concentration-dependent increase in filipin-asso...

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Enrichment of Mammalian Tissues and Xenopus Oocytes with Cholesterol

Two methods of cholesterol enrichment are presented: the application of cyclodextrin saturated with cholesterol to enrich mammalian tissues and cells, and the use of cholesterol-enriched phospholipid-based dispersions (liposomes) to enrich Xenopus oocytes. These methods are instrumental for determining the impact of elevated cholesterol levels in molecular, cellular, and organ function.

Enrichment of Mammalian Tissues and Xenopus Oocytes with Cholesterol

Cholesterol enrichment of mammalian tissues and cells, including Xenopus oocytes used for studying cell function, can be accomplished using a variety of ...

enrichment-of-mammalian-tissues-and-xenopus-oocytes-with-cholesterol

Research

JoVE Journal

Biochemistry

5.9K Views.  The University of Tennessee HSC. Verhoogde cholesterol is een belangrijke risicofactor voor cardiovasculaire en neurodegeneratieve ziekte. Onze protocollen bieden waardevolle instrumenten voor het bestuderen van zowel fysiologische als mechanistische gevolgen van hypercholesterolemie. Deze procedures kunnen worden uitgevoerd met behulp van basislabapparatuur en zijn van toepassing op cellen, weefsels en Xenopus-eicellen.Cholesterol is een belangrijk onderdeel van cellulaire membranen door het hele lichaam. Deze techni...

Video: Enrichment of Mammalian Tissues and Xenopus Oocytes with Cholesterol

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during oogenesis. In order to study roles of such maternal factors in early embryonic development, it is desirable to manipulate their functions from the very beginning of embryonic development. Conventional ways of gene interference are achieved by injection of antisense oligonucleotides (oligos) or mRNA into fertilized eggs, enabling under- or over-expression of specific proteins, respectively. However, these methods normally require more than several hours until protein expression is affected, and, hence, the interference of gene functions is not effective during early embryonic stages. Here, we introduce an experimental system in which expression levels of maternal proteins can be altered before fertilization. Xenopus laevis oocytes obtained from ovaries are defolliculated by incubating with enzymes. Antisense oligos or mRNAs are injected into defolliculated oocytes at the germinal vesicle (GV) stage. These oocytes are in vitro matured to eggs at the metaphase II (MII) stage, followed by intracytoplasmic sperm injection (ICSI). By this way, up to 10% of ICSI embryos can reach the swimming tadpole stage, thus allowing functional tests of specific gene knockdown or overexpression. This approach can be a useful way to study roles of maternally stored factors in early embryonic development.

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

We describe methods of manipulating Xenopus laevis immature oocytes, in vitro maturation of oocytes to eggs, and intracytoplasmic sperm injection. This protocol allows degradation of some maternal proteins and overexpression of genes of interest at fertilization, and hence is valuable to study roles of specific factors in early embryonic development.

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during ...

Developmental Biology

Functional Cloning Using a Xenopus Oocyte Expression System

Identification of genes responsible for embryonic induction poses a number of challenges; to name a few, secreted molecules of interest may be low in abundance, may not be secreted but tethered to the signaling cell(s), or may require the presence of binding partners or upstream regulatory molecules. Thus in a search for gene products capable of eliciting an early lens-inductive response in competent ectoderm, we utilized an expression cloning system that would allow identification of paracrine or juxtacrine factors as well as transcriptional or other regulatory proteins. Pools of mRNA were injected into Xenopus oocytes, and responding tissue placed directly on the oocytes and co-cultured. Following functional cloning of ldb1 from a neural plate stage cDNA library based on its ability to elicit the expression of the early lens placode marker foxe3 in lens-competent animal cap ectoderm, we characterized the mRNA expression pattern, and assayed developmental progression following overexpression or knockdown of ldb1. This system is suitable in a very wide variety of contexts where identification of an inducer or its upstream regulatory molecules is sought using a functional response in competent tissue.

Functional Cloning Using a Xenopus Oocyte Expression System

We describe a Xenopus oocyte and animal cap system for the expression cloning of genes capable of inducing a response in competent ectoderm, and discuss techniques for the subsequent analysis of such genes. This system is useful in the functional identification of a wide range of gene products.

Identification of genes responsible for embryonic induction poses a number of challenges; to name a few, secreted molecules of interest may be low in ...

Cholesterol Efflux Assay

Cholesterol content of cells must be maintained within the very tight limits, too much or too little cholesterol in a cell results in disruption of cellular membranes, apoptosis and necrosis 1. Cells can source cholesterol from intracellular synthesis and from plasma lipoproteins, both sources are sufficient to fully satisfy cells' requirements for cholesterol. The processes of cholesterol synthesis and uptake are tightly regulated and deficiencies of cholesterol are rare 2. Excessive cholesterol is more common problem 3. With the exception of hepatocytes and to some degree adrenocortical cells, cells are unable to degrade cholesterol. Cells have two options to reduce their cholesterol content: to convert cholesterol into cholesteryl esters, an option with limited capacity as overloading cells with cholesteryl esters is also toxic, and cholesterol efflux, an option with potentially unlimited capacity. Cholesterol efflux is a specific process that is regulated by a number of intracellular transporters, such as ATP binding cassette transporter proteins A1 (ABCA1) and G1 (ABCG1) and scavenger receptor type B1. The natural acceptor of cholesterol in plasma is high density lipoprotein (HDL) and apolipoprotein A-I. 
The cholesterol efflux assay is designed to quantitate the rate of cholesterol efflux from cultured cells. It measures the capacity of cells to maintain cholesterol efflux and/or the capacity of plasma acceptors to accept cholesterol released from cells. The assay consists of the following steps. Step 1: labelling cellular cholesterol by adding labelled cholesterol to serum-containing medium and incubating with cells for 24-48 h. This step may be combined with loading of cells with cholesterol. Step 2: incubation of cells in serum-free medium to equilibrate labelled cholesterol among all intracellular cholesterol pools. This stage may be combined with activation of cellular cholesterol transporters. Step 3: incubation of cells with extracellular acceptor and quantitation of movement of labelled cholesterol from cells to the acceptor. If cholesterol precursors were used to label newly synthesized cholesterol, a fourth step, purification of cholesterol, may be required.
The assay delivers the following information: (i) how a particular treatment (a mutation, a knock-down, an overexpression or a treatment) affects the capacity of cell to efflux cholesterol and (ii) how the capacity of plasma acceptors to accept cholesterol is affected by a disease or a treatment. This method is often used in context of cardiovascular research, metabolic and neurodegenerative disorders, infectious and reproductive diseases.

The cholesterol assay is designed to quantitate the rate of cholesterol efflux from cultured cells and the capacity of plasma acceptors to accept cholesterol released from cells. The assay consists of labelling cells with cholesterol, equilibration of cholesterol among intracellular pools and release of cholesterol to an extracellular acceptor.

Cholesterol content of cells must be maintained within the very tight limits, too much or too little cholesterol in a cell results in disruption of ...

Medicine

Reconstitution of Cell-cycle Oscillations in Microemulsions of Cell-free Xenopus Egg Extracts

Real-time measurement of oscillations at the single-cell level is important to uncover the mechanisms of biological clocks. Although bulk extracts prepared from Xenopus laevis eggs have been powerful in dissecting biochemical networks underlying the cell-cycle progression, their ensemble average measurement typically leads to a damped oscillation, despite each individual oscillator being sustained. This is due to the difficulty of perfect synchronization among individual oscillators in noisy biological systems. To retrieve the single-cell dynamics of the oscillator, we developed a droplet-based artificial cell system that can reconstitute mitotic cycles in cell-like compartments encapsulating cycling cytoplasmic extracts of Xenopus laevis eggs. These simple cytoplasmic-only cells exhibit sustained oscillations for over 30 cycles. To build more complicated cells with nuclei, we added demembranated sperm chromatin to trigger nuclei self-assembly in the system. We observed a periodic progression of chromosome condensation/decondensation and nuclei envelop breakdown/reformation, like in real cells. This indicates that the mitotic oscillator functions faithfully to drive multiple downstream mitotic events. We simultaneously tracked the dynamics of the mitotic oscillator and downstream processes in individual droplets using multi-channel time-lapse fluorescence microscopy. The artificial cell-cycle system provides a high-throughput framework for quantitative manipulation and analysis of mitotic oscillations with single-cell resolution, which likely provides important insights into the regulatory machinery and functions of the clock.

Reconstitution of Cell-cycle Oscillations in Microemulsions of Cell-free Xenopus Egg Extracts

We present a method for the generation of in vitro self-sustained mitotic oscillations at the single-cell level by encapsulating egg extracts of Xenopus laevis in water-in-oil microemulsions.

Real-time measurement of oscillations at the single-cell level is important to uncover the mechanisms of biological clocks. Although bulk extracts ...

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development

Kinase activity is crucial for a plethora of cellular functions, including cell proliferation, differentiation, migration, and apoptosis. During early embryonic development, kinase activity is highly dynamic and widespread across the embryo. Pharmacological and genetic approaches are commonly used to probe kinase activities. Unfortunately, it is challenging to achieve superior spatial and temporal resolution using these strategies. Furthermore, it is not feasible to control the kinase activity in a reversible fashion in live cells and multicellular organisms. Such a limitation remains a bottleneck for achieving a quantitative understanding of kinase activity during development and differentiation. This work presents an optogenetic strategy that takes advantage of a bicistronic system containing photoactivatable proteins Arabidopsis thaliana cryptochrome 2 (CRY2) and the N-terminal domain of cryptochrome-interacting basic-helix-loop-helix (CIBN). Reversible activation of the mitogen-activated protein kinase (MAPK) signaling pathway is achieved through light-mediated protein translocation in live cells. This approach can be applied to mammalian cell cultures and live vertebrate embryos. This bicistronic system can be generalized to control the activity of other kinases with similar activation mechanisms and can be applied to other model systems.

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development

This protocol describes an optogenetic strategy to modulate mitogen-activated protein kinase (MAPK) activity during cell differentiation and Xenopus embryonic development. This method allows for the reversible activation of the MAPK signaling pathway in mammalian cell culture and in multicellular live organisms, like Xenopus embryos, with high spatial and temporal resolution.

Kinase activity is crucial for a plethora of cellular functions, including cell proliferation, differentiation, migration, and apoptosis. During early ...

Combinational Treatment of Trichostatin A and Vitamin C Improves the Efficiency of Cloning Mice by Somatic Cell Nuclear Transfer

Somatic cell nuclear transfer (SCNT) provides a unique opportunity to directly produce a cloned animal from a donor cell, and it requires the use of skillful techniques. Additionally, the efficiencies of cloning have remained low since the successful production of cloned animals, especially mice. There have been many attempts to improve the cloning efficiency, and trichostatin A (TSA), a histone deacetylase inhibitor, has been widely used to enhance the efficiency of cloning. Here, we report a dramatically improved cloning method in mice. This somatic cell nuclear transfer method involves usage of Hemagglutinating virus of Japan Envelope (HVJ-E), which enables easy manipulation. Moreover, the treatment using two small molecules, TSA and vitamin C (VC), with deionized bovine serum albumin (dBSA), is highly effective for embryonic development. This approach requires neither additional injection nor genetic manipulation, and thus presents a simple, suitable method for practical use. This method could become a technically feasible approach for researchers to produce genetically modified animals from cultured cells. Furthermore, it might be a useful way for the rescue of endangered animals via cloning.

We describe a dramatically improved method for mouse cloning using trichostatin A, vitamin C, and deionized bovine serum albumin. We show a simplified, reproducible protocol that supports efficient development of cloned embryos. Hence, this method could become a standardized procedure for mouse cloning.

Somatic cell nuclear transfer (SCNT) provides a unique opportunity to directly produce a cloned animal from a donor cell, and it requires the use of ...

Fertilization of Xenopus oocytes using the Host Transfer Method

Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to generate maternal-effect mutant animals. Additionally, many of the techniques to overexpress or inhibit gene function in organisms such as Xenopus and zebrafish fail to sufficiently target critical maternal signaling pathways, such as Wnt signaling. In Xenopus, manipulating gene function in cultured oocytes and subsequently fertilizing them can ameliorate these problems to some extent. Oocytes are manually defolliculated from donor ovary tissue, injected or treated in culture as desired, and then stimulated with progesterone to induce maturation. Next, the oocytes are introduced into the body cavity of an ovulating host female frog, whereupon they will be translocated through the host's oviduct and acquire modifications and jelly coats necessary for fertilization. The resulting embryos can then be raised to the desired stage and analyzed for the effects of any experimental perturbations. This host-transfer method has been highly effective in uncovering basic mechanisms of early development and allows a wide range of experimental possibilities not available in any other vertebrate model organism.

Fertilization of Xenopus oocytes using the Host Transfer Method

Procedure for fertilizing Xenopus oocytes by the host transfer method.

Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to ...

Biology

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e. "free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions.

Here we describe a method to visualize endoplasmic reticulum-associated mRNAs in mammalian tissue culture cells. This technique involves the selective permeabilization of the plasma membrane with digitonin to remove cytoplasmic contents followed by fluorescent in situ hybridization to detect either bulk poly(A) mRNA or specific transcripts.

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

High-throughput Nitrobenzoxadiazole-labeled Cholesterol Efflux Assay

Atherosclerosis leads to cardiovascular disease (CVD). It is still unclear whether cholesterol-HDL (cHDL) concentration plays a causal role in atherosclerosis development. However, an important factor in early stages of atheroma plaque formation is cholesterol efflux capacity to HDL (the ability of HDL particles to accept cholesterol from macrophages) in order to avoid foam cell formation. This is a key step in avoiding the accumulation of cholesterol in the endothelium and a part of reverse cholesterol transport (RCT) to eliminate cholesterol through the liver. Cholesterol efflux capacity to serum or plasma in macrophage cell models is a promising tool that can be used as biomarker for atherosclerosis. Traditionally, [3H]-cholesterol has been used in cholesterol efflux assays. In this study, we aim to develop a safer and faster strategy using fluorescent labelled-cholesterol (NBD-cholesterol) in a cellular assay to trace the cholesterol uptake and efflux process in THP-1-derived macrophages. Finally, we optimize and standardize the NBD-cholesterol efflux method and develop a high-throughput analysis using 96-well plates.

Measurement of in vitro cholesterol efflux capacity to serum or plasma in macrophage cell models is a promising tool as a biomarker for atherosclerosis. In the present study, we optimize and standardize a fluorescent NBD-cholesterol efflux method and develop a high-throughput analysis using 96-well plates.

Atherosclerosis leads to cardiovascular disease (CVD). It is still unclear whether cholesterol-HDL (cHDL) concentration plays a causal role in ...

Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments

The Xenopus oocyte as a heterologous expression system for proteins, was first described by Gurdon et al.1 and has been widely used since its discovery (References 2 - 3, and references therein). A characteristic that makes the oocyte attractive for foreign channel expression is the poor abundance of endogenous ion channels4. This expression system has proven useful for the characterization of many proteins, among them ligand-gated ion channels.
The expression of GABAA receptors in Xenopus oocytes and their functional characterization is described here, including the isolation of oocytes, microinjections with cRNA, the removal of follicular cell layers, and fast solution changes in electrophysiological experiments. The procedures were optimized in this laboratory5,6 and deviate from the ones routinely used7-9. Traditionally, denuded oocytes are prepared with a prolonged collagenase treatment of ovary lobes at RT, and these denuded oocytes are microinjected with mRNA. Using the optimized methods, diverse membrane proteins have been expressed and studied with this system, such as recombinant GABAA receptors10-12, human recombinant chloride channels13, Trypanosome potassium channels14, and a myo-inositol transporter15, 16.
The methods detailed here may be applied to the expression of any protein of choice in Xenopus oocytes, and the rapid solution change can be used to study other ligand-gated ion channels.

Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments

Optimized procedures for the isolation of single follicles, cytoplasmic RNA microinjections, the removal of surrounding cell layers, and protein expression in Xenopus oocytes are described. In addition, a simple method for fast solution changes in electrophysiological experiments with ligand-gated ion channels is presented.

The Xenopus oocyte as a heterologous expression system for proteins, was first described by Gurdon et al.1 and has been widely used since its discovery ...

Enrichment of Mammalian Tissues and Xenopus Oocytes with Cholesterol

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