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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>MgCl2</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>KH2PO4</td>
			<td>Fisher</td>
			<td>P285</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</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>NaHCO3</td>
			<td>Sigma</td>
			<td>S6014</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</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&#8482; 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&#8482; BOD Refrigerated Incubator</td>
			<td>FIsher</td>
			<td>97-990E</td>
			<td></td>
		</tr>
		<tr>
			<td>Oocytes</td>
			<td>Xenoocyte&#8482;</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-&#946;-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 &#176;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 H2O</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 &#956;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...

Watch this Scientific Journal Video about Enrichment of Mammalian Tissues and Xenopus Oocytes with Cholesterol at JoVE.com

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

5.8K Views.  The University of Tennessee HSC. 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.

Video: Enrichment of Mammalian Tissues and Xenopus Oocytes with Cholesterol

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this technique is also frequently referred to as immunoprecipitation. Affinity capture can be applied in a bench-scale and in a high-throughput context. When coupled with protein mass spectrometry, affinity capture has proven to be a workhorse of interactome analysis. Although there are potentially many ways to execute the numerous steps involved, the following protocols implement our favored methods. Two features are distinctive: the use of cryomilled cell powder to produce cell extracts, and antibody-coupled paramagnetic beads as the affinity medium. In many cases, we have obtained superior results to those obtained with more conventional affinity capture practices. Cryomilling avoids numerous problems associated with other forms of cell breakage. It provides efficient breakage of the material, while avoiding denaturation issues associated with heating or foaming. It retains the native protein concentration up to the point of extraction, mitigating macromolecular dissociation. It reduces the time extracted proteins spend in solution, limiting deleterious enzymatic activities, and it may reduce the non-specific adsorption of proteins by the affinity medium. Micron-scale magnetic affinity media have become more commonplace over the last several years, increasingly replacing the traditional agarose- and Sepharose-based media. Primary benefits of magnetic media include typically lower non-specific protein adsorption; no size exclusion limit because protein complex binding occurs on the bead surface rather than within pores; and ease of manipulation and handling using magnets.

Here we describe protocols to disrupt mammalian cells by solid-state milling at a cryogenic temperature, produce a cell extract from the resulting cell powder, and isolate protein complexes of interest by affinity capture upon antibody-coupled micron-scale paramagnetic beads.

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this ...

Voltage-clamp Fluorometry in Xenopus Oocytes Using Fluorescent Unnatural Amino Acids

Voltage-Clamp Fluorometry (VCF) has been the technique of choice to investigate the structure and function of electrogenic membrane proteins where real-time measurements of fluorescence and currents simultaneously report on local rearrangements and global function, respectively1. While high-resolution structural techniques such as cryo-electron microscopy or X-ray crystallography provide static images of the proteins of interest, VCF provides dynamic structural data that allows us to link the structural rearrangements (fluorescence) to dynamic functional data (electrophysiology). Until recently, the thiol-reactive chemistry used for site-directed fluorescent labeling of the proteins restricted the scope of the approach because all accessible cysteines, including endogenous ones, will be labeled. It was thus required to construct proteins free of endogenous cysteines. Labeling was also restricted to sites accessible from the extracellular side. This changed with the use of Fluorescent Unnatural Amino Acids (fUAA) to specifically incorporate a small fluorescent probe in response to stop codon suppression using an orthogonal tRNA and tRNA synthetase pair2. The VCF improvement only requires a two-step injection procedure of DNA injection (tRNA/synthetase pair) followed by RNA/fUAA co-injection. Now, labelling both intracellular and buried sites is possible, and the use of VCF has expanded significantly. The VCF technique thereby becomes attractive for studying a wide range of proteins and &#8211; more importantly &#8211; allows investigating numerous cytosolic regulatory mechanisms.

Voltage-clamp Fluorometry in Xenopus Oocytes Using Fluorescent Unnatural Amino Acids

This article describes an enhancement of conventional Voltage-Clamp Fluorometry (VCF) where Fluorescent Unnatural Amino Acids (fUAA) are used instead of maleimide dyes, to probe structural rearrangements in ion channels. The procedure includes Xenopus oocyte DNA injection, RNA/fUAA coinjection, and simultaneous current and fluorescence measurements.

Voltage-Clamp Fluorometry (VCF) has been the technique of choice to investigate the structure and function of electrogenic membrane proteins where ...

Enrichment of Detergent-insoluble Protein Aggregates from Human Postmortem Brain

In this study, we describe an abbreviated single-step fractionation protocol for the enrichment of detergent-insoluble protein aggregates from human postmortem brain. The ionic detergent N-lauryl-sarcosine (sarkosyl) effectively solubilizes natively folded proteins in brain tissue allowing the enrichment of detergent-insoluble protein aggregates from a wide range of neurodegenerative proteinopathies, such as Alzheimer&#39;s disease (AD), Parkinson&#39;s disease and amyotrophic lateral sclerosis, and prion diseases. Human control and AD postmortem brain tissues were homogenized and sedimented by ultracentrifugation in the presence of sarkosyl to enrich detergent-insoluble protein aggregates including pathologic phosphorylated tau, the core component of neurofibrillary tangles in AD. Western blotting demonstrated the differential solubility of aggregated phosphorylated-tau and the detergent-soluble protein, Early Endosome Antigen 1 (EEA1) in control and AD brain. Proteomic analysis also revealed enrichment of &#946;-amyloid (A&#946;), tau, snRNP70 (U1-70K), and apolipoprotein E (APOE) in the sarkosyl-insoluble fractions of AD brain compared to those of control, consistent with previous tissue fractionation strategies. Thus, this simple enrichment protocol is ideal for a wide range of experimental applications ranging from Western blotting and functional protein co-aggregation assays to mass spectrometry-based proteomics.

An abbreviated fractionation protocol for the enrichment of detergent-insoluble protein aggregates from human postmortem brain is described.

In this study, we describe an abbreviated single-step fractionation protocol for the enrichment of detergent-insoluble protein aggregates from human ...

Imaging Amyloid Tissues Stained with Luminescent Conjugated Oligothiophenes by Hyperspectral Confocal Microscopy and Fluorescence Lifetime Imaging

Proteins that deposit as amyloid in tissues throughout the body can be the cause or consequence of a large number of diseases. Among these we find neurodegenerative diseases such as Alzheimer&#39;s and Parkinson&#39;s disease afflicting primarily the central nervous system, and systemic amyloidosis where serum amyloid A, transthyretin and IgG light chains deposit as amyloid in liver, carpal tunnel, spleen, kidney, heart, and other peripheral tissues. Amyloid has been known and studied for more than a century, often using amyloid specific dyes such as Congo red and Thioflavin T (ThT) or Thioflavin (ThS). In this paper, we present heptamer-formyl thiophene acetic acid (hFTAA) as an example of recently developed complements to these dyes called luminescent conjugated oligothiophenes (LCOs). hFTAA is easy to use and is compatible with co-staining in immunofluorescence or with other cellular markers. Extensive research has proven that hFTAA detects a wider range of disease associated protein aggregates than conventional amyloid dyes. In addition, hFTAA can also be applied for optical assignment of distinct aggregated morphotypes to allow studies of amyloid fibril polymorphism. While the imaging methodology applied is optional, we here demonstrate hyperspectral imaging (HIS), laser scanning confocal microscopy and fluorescence lifetime imaging (FLIM). These examples show some of the imaging techniques where LCOs can be used as tools to gain more detailed knowledge of the formation and structural properties of amyloids. An important limitation to the technique is, as for all conventional optical microscopy techniques, the requirement for microscopic size of aggregates to allow detection. Furthermore, the aggregate should comprise a repetitive &#946;-sheet structure to allow for hFTAA binding. Excessive fixation and/or epitope exposure that modify the aggregate structure or conformation can render poor hFTAA binding and hence pose limitations to accurate imaging.

Amyloid deposition is a hallmark of different diseases and afflicts many different organs. This paper describes the application of luminescent conjugated oligothiophene fluorescence staining in combination with fluorescence microscopy techniques. This staining method represents a powerful tool for detection and exploration of protein aggregates in both clinical and scientific setups.

Proteins that deposit as amyloid in tissues throughout the body can be the cause or consequence of a large number of diseases. Among these we find ...

Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation of protein synthesis is the key to cellular stress-response, and dysregulation is central to many disease states, such as cancer. For instance, although cellular stress leads to the inhibition of global translation by attenuating cap-dependent initiation, certain stress-response proteins are selectively translated in a cap-independent manner. Discreet RNA regulatory elements, such as cellular internal ribosome entry sites (IRESes), allow for the translation of these specific mRNAs. Identification of such mRNAs, and the characterization of their regulatory mechanisms, have been a key area in molecular biology. Toeprinting is a method for the study of RNA structure and function as it pertains to translation initiation. The goal of toeprinting is to assess the ability of in vitro transcribed RNA to form stable complexes with ribosomes under a variety of conditions, in order to determine which sequences, structural elements, or accessory factors are involved in ribosome binding&#8212;a pre-cursor for efficient translation initiation. Alongside other techniques, such as western analysis and polysome profiling, toeprinting allows for a robust characterization of mechanisms for the regulation of translation initiation.

Toeprinting aims to measure the ability of in vitro transcribed RNA to form translation initiation complexes with ribosomes under a variety of conditions. This protocol describes a method for toeprinting mammalian RNA and can be used to study both cap-dependent and IRES-driven translation.

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation ...

Expression and Purification of Mammalian Bestrophin Ion Channels

The human genome encodes four bestrophin paralogs, namely BEST1, BEST2, BEST3, and BEST4. BEST1, encoded by the BEST1 gene, is a Ca2+-activated Cl- channel (CaCC) predominantly expressed in retinal pigment epithelium (RPE). The physiological and pathological significance of BEST1 is highlighted by the fact that over 200 distinct mutations in the BEST1 gene have been genetically linked to a spectrum of at least five retinal degenerative disorders, such as Best vitelliform macular dystrophy (Best disease). Therefore, understanding the biophysics of bestrophin channels at the single-molecule level holds tremendous significance. However, obtaining purified mammalian ion channels is often a challenging task. Here, we report a protocol for the expression of mammalian bestrophin proteins with the BacMam baculovirus gene transfer system and their purification by affinity and size-exclusion chromatography. The purified proteins have the potential to be utilized in subsequent functional and structural analyses, such as electrophysiological recording in lipid bilayers and crystallography. Importantly, this pipeline can be adapted to study the functions and structures of other ion channels.

The purification of ion channels is often challenging, but once achieved, it can potentially allow in vitro investigations of the functions and structures of the channels. Here, we describe the stepwise procedures for the expression and purification of mammalian bestrophin proteins, a family of Ca2+-activated Cl- channels.

The human genome encodes four bestrophin paralogs, namely BEST1, BEST2, BEST3, and BEST4. BEST1, encoded by the BEST1 gene, is a Ca2+-activated Cl- ...

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ubiquitinated proteins, peptides with a diglycine remnant conjugated to the epsilon amino group of lysine (&#39;K-&#949;-diglycine&#39; or simply &#39;diGly&#39;) can be used to track back the original modification site. Efficient immunopurification of diGly peptides combined with sensitive detection by mass spectrometry has resulted in a huge increase in the number of ubiquitination sites identified up to date. We have made several improvements to this workflow, including offline high pH reverse-phase fractionation of peptides prior to the enrichment procedure, and the inclusion of more advanced peptide fragmentation settings in the ion routing multipole. Also, more efficient cleanup of the sample using a filter-based plug in order to retain the antibody beads results in a greater specificity for diGly peptides. These improvements result in the routine detection of more than 23,000 diGly peptides from human cervical cancer cells (HeLa) cell lysates upon proteasome inhibition in the cell. We show the efficacy of this strategy for in-depth analysis of the ubiquitinome profiles of several different cell types and of in vivo samples, such as brain tissue. This study presents an original addition to the toolbox for protein ubiquitination analysis to uncover the deep cellular ubiquitinome.

We present a method for the purification, detection, and identification of diGly peptides that originate from ubiquitinated proteins from complex biological samples. The presented method is reproducible, robust, and outperforms published methods with respect to the level of depth of the ubiquitinome analysis.

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ...

Studying RNA Interactors of Protein Kinase RNA-Activated during the Mammalian Cell Cycle

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral RNAs. When bound to viral double-stranded RNAs (dsRNAs), PKR undergoes dimerization and subsequent autophosphorylation. Phosphorylated PKR (pPKR) becomes active and induces phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2&#945;) to suppress global translation. Increasing evidence suggests that PKR can be activated under physiological conditions such as during the cell cycle or under various stress conditions without infection. However, our understanding of the RNA activators of PKR is limited due to the lack of a standardized experimental method to capture and analyze PKR-interacting dsRNAs. Here, we present an experimental protocol to specifically enrich and analyze PKR bound RNAs during the cell cycle using HeLa cells. We utilize the efficient crosslinking activity of formaldehyde to fix PKR-RNA complexes and isolate them via immunoprecipitation. PKR co-immunoprecipitated RNAs can then be further processed to generate a high-throughput sequencing library. One major class of PKR-interacting cellular dsRNAs is mitochondrial RNAs (mtRNAs), which can exist as intermolecular dsRNAs through complementary interaction between the heavy-strand and the light-strand RNAs. To study the strandedness of these duplex mtRNAs, we also present a protocol for strand-specific qRT-PCR. Our protocol is optimized for the analysis of PKR-bound RNAs, but it can be easily modified to study cellular dsRNAs or RNA-interactors of other dsRNA binding proteins.

We present experimental approaches for studying RNA-interactors of double-stranded RNA binding protein kinase RNA-activated (PKR) during the mammalian cell cycle using HeLa cells. This method utilizes formaldehyde to crosslink RNA-PKR complexes and immunoprecipitation to enrich PKR-bound RNAs. These RNAs can be further analyzed through high-throughput sequencing or qRT-PCR.

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral ...

Enrichment of Native Lipoprotein Particles with microRNA and Subsequent Determination of Their Absolute/Relative microRNA Content and Their Cellular Transfer Rate

Lipoprotein particles are predominately transporters of lipids and cholesterol in the bloodstream. Furthermore, they contain small amounts of strands of noncoding microRNA (miRNA). In general, miRNA alters the protein expression profile due to interactions with messenger-RNA (mRNA). Thus, knowledge of the relative and absolute miRNA content of lipoprotein particles is essential to estimate the biological effect of cellular particle uptake. Here, a quantitative real-time polymerase chain reaction (qPCR)-based protocol is presented to determine the absolute miRNA content of lipoprotein particles&#8212;exemplified shown for native and miRNA-enriched lipoprotein particles. The relative miRNA content is quantified using multiwell microfluidic array cards. Furthermore, this protocol allows scientists to estimate the cellular miRNA and, thus, the lipoprotein particle uptake rate. A significant increase of the cellular miRNA level is observable when using high-density lipoprotein (HDL) particles artificially loaded with miRNA, whereas incubation with native HDL particles yields no significant effect due to their rather low miRNA content. In contrast, the cellular uptake of low-density lipoprotein (LDL) particles&#8212;neither with native miRNA nor artificially loaded with it&#8212;did not alter the cellular miRNA level.

Here, a quantitative real-time polymerase chain reaction-based protocol is presented for the determination of the native micro-RNA content (absolute/relative) of lipoprotein particles. In addition, a method for increasing the micro-RNA level, as well as a method for determining the cellular uptake rate of lipoprotein particles, is demonstrated.

Lipoprotein particles are predominately transporters of lipids and cholesterol in the bloodstream. Furthermore, they contain small amounts of strands of ...

Quantification of Coenzyme A in Cells and Tissues

Emerging research has revealed that the cellular coenzyme A (CoA) supply can become limiting with a detrimental impact on growth, metabolism and survival. Measurement of cellular CoA is a challenge due to its relatively low abundance and the dynamic conversion of free CoA to CoA thioesters that, in turn, participate in numerous metabolic reactions. A method is described that navigates through potential pitfalls during sample preparation to yield an assay with a broad linear range of detection that is suitable for use in many biomedical laboratories.

This method describes sample preparation from cultured cells and animal tissues, extraction and derivatization of coenzyme A in the samples, followed by high pressure liquid chromatography for purification and quantification of the derivatized coenzyme A by absorbance or fluorescence detection.

Emerging research has revealed that the cellular coenzyme A (CoA) supply can become limiting with a detrimental impact on growth, metabolism and survival. ...