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Cholesterol methyl β cyclodextrin

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Cholesterol-methyl-β-cyclodextrin is a laboratory reagent used to study cholesterol-related biological processes. It acts as a solubilizing agent for cholesterol, improving its solubility in aqueous solutions. This property makes it a useful tool for researchers investigating cholesterol metabolism, transport, and related cellular functions.

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Lab products found in correlation

Cerulenin, by Merck Group (2 mentions) Etomoxir, by Merck Group (2 mentions) Allopurinol, by Merck Group (2 mentions) Palmitic, by Cayman Chemical (2 mentions) Oleic acid, by Cayman Chemical (2 mentions) Simvastatin, by Merck Group (2 mentions) Mk 2206, by Cayman Chemical (2 mentions) Af 214 14, by Thermo Fisher Scientific (2 mentions) 25 hydroxycholesterol, by Cayman Chemical (2 mentions) Diphenyleneiodonium chloride dpi, by Merck Group (2 mentions) L ng nitroarginine methyl ester l name, by Cayman Chemical (2 mentions) Sirt1 activator 2, by Merck Group (2 mentions) Ex 527, by Merck Group (2 mentions) Aicar, by Abcam (2 mentions) Hmgcoa, by Merck Group (2 mentions) D4902, by Merck Group (2 mentions) Compound c, by Merck Group (2 mentions) N acetylcysteine, by Merck Group (2 mentions) Hydrogen peroxide, by Merck Group (2 mentions) Electron microscopy reagents, by Electron Microscopy Sciences (2 mentions)

16 protocols using cholesterol methyl β cyclodextrin

1

Polarization of Bone Marrow-Derived Macrophages

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BMMФ were stimulated either with IL-4 (10 ng/ml, AF-214-14, Peprotech), lipopolysaccharide (100 ng/ml, Sigma) or dexamethasone (100nM, D4902, Sigma) to respectively achieve M(IL-4), M(LPS) or M(GC) polarization state. In some experiments, BMMФ were treated with C75 (Sigma), Cerulenin (Sigma), Etomoxir (Sigma), pan-AKT inhibitor MK-2206 (1 mM, Cayman Chemical), 25-hydroxycholesterol (10 μM, Cayman chemical), N-acetyl cysteine (10mM, Sigma), Diphenyleneiodonium chloride (DPI, 5 μM, Sigma), L-NG-Nitroarginine methyl ester (L-NAME, 1mM, Cayman Chemical), Allopurinol (100 μM, Sigma), hydrogen peroxide (Sigma), SIRT1 activator II (Sigma), EX-527 (Sigma), Compound C (Sigma), AICAR (Abcam), HMGCoA (Sigma), water-soluble cholesterol (Cholesterol–methyl-β-cyclodextrin, Sigma) or Simvastatin (Sigma).
For fatty acid treatment, palmitic or oleic acid (Cayman) were solubilized at 100mM in absolute ethanol at 60°C. Fatty acid were conjugated at the desired concentration using a sonicator water bath into the macrophage culture medium to avoid endotoxin contamination from BSA54 (link).
Bidault G., Virtue S., Petkevicius K., Jolin H.E., Dugourd A., Guénantin A.C., Leggat J., Mahler-Araujo B., Lam B.Y., Ma M.K., Dale M., Carobbio S., Kaser A., Fallon P.G., Saez-Rodriguez J., McKenzie A.N, & Vidal-Puig A. (2021). SREBP1-induced fatty acid synthesis depletes macrophages antioxidant defences to promote their alternative activation. Nature metabolism, 3(9), 1150-1162.
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2

Cholesterol Metabolism Assay

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Cholesterol, 25OHC, and 7α,25OHC were purchased from Sigma-Aldrich (St. Louis, MI, USA) or Avanti Polar Lipids (Alabaster, AL, USA), resolved in DMSO, and stored at −80°C until use. Hexane, ethylacetate, and sodium sulfate anhydrous were purchased from Nacalai Tesque (Kyoto, Japan). 25-hydroxyCholesterol and D6-25-hydroxyCholesterol were obtained from Avanti Polar Lipids. Cholesterol-methyl-β-cyclodextrin was purchased from Sigma-Aldrich.
Takahashi H., Nomura H., Iriki H., Kubo A., Isami K., Mikami Y., Mukai M., Sasaki T., Yamagami J., Kudoh J., Ito H., Kamata A., Kurebayashi Y., Yoshida H., Yoshimura A., Sun H.W., Suematsu M., O’Shea J.J., Kanno Y, & Amagai M. (2021). Cholesterol 25-hydroxylase is a metabolic switch to constrain T cell-mediated inflammation in the skin. Science immunology, 6(64), eabb6444.
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3

Cholesterol Loading of Aortic SMCs

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MOVAS, which is a commonly used mouse aortic SMC line, and human primary aortic SMCs were purchased from ATCC (Manassas, VA, USA). The cells were cultured in DMEM high-glucose full medium (catalog no. [cat.] 11965092; ThermoFisher) supplemented with 10% FBS and 50 μg/mL G418 (antibiotic, cat. 4727878001; Sigma-Aldrich) at 5% CO2 and 37°C. Primary rat aortic SMCs were isolated as described previously42 (link) with minor modifications based on a recent report.50 (link) The cells were cultured in smooth muscle complete (full) medium (cat. M2268; Cell Biologics) and used at passage 5. To load cholesterol into SMCs, we followed a previously reported method using a soluble form of cholesterol, i.e., Cholesterol-methyl-β-cyclodextrin (cat. C4951; Sigma-Aldrich).1 (link),2 (link) Cholesterol-methyl-β-cyclodextrin (concentration indicated in the figure legends) in 0.25% (w/v) BSA was added to subconfluent SMCs and incubated for 72 h prior to harvest for various assays.2 (link),27 (link) No obvious apoptosis was observed after cholesterol loading (Figure S1B). The cells incubated with 0.25% BSA for 72 h without Cholesterol-methyl-β-cyclodextrin served as solvent controls.2 (link)
Li J., Shen H., Owens G.K, & Guo L.W. (2022). SREBP1 regulates Lgals3 activation in response to cholesterol loading. Molecular Therapy. Nucleic Acids, 28, 892-909.
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4

Quantitative Cholesterol Extraction and Reloading

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Cells were seeded at 1 × 105 cells/well in a six-well plate 48 h prior to cholesterol extraction. Cells were washed briefly with cold PBS (twice), followed by addition of 1 mL of hexane:isopropanol (3:2) to the wells for lipid extraction, followed by incubation at room temperature for 30 min. The lipid-containing mixture was recovered in an Eppendorf tube and air-dried using an Iwaki Halogen Vacuum Concentrator (IVC-500) for 20 min at room temperature. The pellet was then resuspended and cholesterol content was determined using the Amplex RedTM Cholesterol Assay Kit (Invitrogen). For reloading cholesterol, cholesterol-methyl-β-cyclodextrin (C4951, Sigma) was added to the culture medium at the final concentration of 10 μg/mL for 24 h47 (link),48 (link). Incorporation of cholesterol into cells was estimated by adding CholEsteryl BODIPY FL C12 (Thermo Fisher Scientific) to the culture medium (concentration, 5 μM) for 2 h before fixation and immunofluorescence analysis.
Chee W.Y., Kurahashi Y., Kim J., Miura K., Okuzaki D., Ishitani T., Kajiwara K., Nada S., Okano H, & Okada M. (2021). β-catenin-promoted cholesterol metabolism protects against cellular senescence in naked mole-rat cells. Communications Biology, 4, 357.
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5

Lipid Membrane Composition Analysis

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BODIPY-sphingosine was a gift from Robert Bittman (Queens College of CUNY Flushing, NY). Reagents purchased from commercial sources: methyl-β-cyclodextrin, cholesterol-methyl-β-cyclodextrin (cholesterol-water soluble), 2-hydroxypropyl-β-cyclodextrin and sphingomyelinase from Bacillus cereus (Sigma-Aldrich, St. Louis, MO); Dil and BODIPY-TR-ceramide, 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine triethylammonium salt (TR-DHPE)(Life Technologies, Carlsbad, CA); TopFluor PS, brain sphingosine, egg PC, liver PE, cholesterol from sheep wool, brain PS, egg PA, brain PI(4)P, brain PI(4,5)P2, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-2000] (Avanti Polar Lipids, Alabaster, AL); and ATP, [γ-32P] (PerkinElmer, Waltham, MA). Electron microscopy reagents were purchased from Electron Microscopy Sciences.
Shen H., Giordano F., Wu Y., Chan J., Zhu C., Milosevic I., Wu X., Yao K., Chen B., Baumgart T., Sieburth D, & De Camilli P. (2014). Coupling between endocytosis and sphingosine kinase I recruitment. Nature cell biology, 16(7), 652-662.
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6

Cholesterol metabolism in EndoC-βH1 cells

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The human pancreatic beta cell line EndoC-βH1 cells (Univercell Biosolutions [mycoplasma negative]) was cultured as previously described (42 (link)). Experiments were carried out 24 h after seeding the cells at a density of approximately 105 cells/cm2. The following compounds were used for treatment: cholesterol-methyl-β-cyclodextrin (catalog no.: C4951; Sigma–Aldrich), mevastatin (catalog no.: M2537; Sigma–Aldrich), human native LDL and copper-oxidized LDL or VLDL (see later for lipoprotein extraction protocol), human recombinant wildtype PCSK9 (CircuLex; catalog no.: CY-R2330).
Saitoski K., Ryaboshapkina M., Hamza G.M., Jarnuczak A.F., Berthault C., Carlotti F., Armanet M., Sengupta K., Underwood C.R., Andersson S., Guillas I., Le Goff W, & Scharfmann R. (2022). Proprotein convertase PCSK9 affects expression of key surface proteins in human pancreatic beta cells via intracellular and extracellular regulatory circuits. The Journal of Biological Chemistry, 298(7), 102096.
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7

Lipid Signaling Pathway Modulation

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Polyunsaturated fatty acids, deuterated arachidonic acid (AA‐d8), 5,8,11,14‐eicosatetraynoic acid (ETYA), and Triacsin C were obtained from Cayman Chemicals (Hamburg, Germany), Ruxolitinib from InvivoGen, BIRB796 (Doramapimod) from Biomol, SB203580 from Biozol (Eching, Germany), cholesterol‐methyl‐β‐cyclodextrin from Sigma‐Aldrich.
Hammoud M.K., Dietze R., Pesek J., Finkernagel F., Unger A., Bieringer T., Nist A., Stiewe T., Bhagwat A.M., Nockher W.A., Reinartz S., Müller‐Brüsselbach S., Graumann J, & Müller R. (2022). Arachidonic acid, a clinically adverse mediator in the ovarian cancer microenvironment, impairs JAK‐STAT signaling in macrophages by perturbing lipid raft structures. Molecular Oncology, 16(17), 3146-3166.
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8

Lipid Membrane Composition Analysis

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BODIPY-sphingosine was a gift from Robert Bittman (Queens College of CUNY Flushing, NY). Reagents purchased from commercial sources: methyl-β-cyclodextrin, cholesterol-methyl-β-cyclodextrin (cholesterol-water soluble), 2-hydroxypropyl-β-cyclodextrin and sphingomyelinase from Bacillus cereus (Sigma-Aldrich, St. Louis, MO); Dil and BODIPY-TR-ceramide, 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine triethylammonium salt (TR-DHPE)(Life Technologies, Carlsbad, CA); TopFluor PS, brain sphingosine, egg PC, liver PE, cholesterol from sheep wool, brain PS, egg PA, brain PI(4)P, brain PI(4,5)P2, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-2000] (Avanti Polar Lipids, Alabaster, AL); and ATP, [γ-32P] (PerkinElmer, Waltham, MA). Electron microscopy reagents were purchased from Electron Microscopy Sciences.
Shen H., Giordano F., Wu Y., Chan J., Zhu C., Milosevic I., Wu X., Yao K., Chen B., Baumgart T., Sieburth D, & De Camilli P. (2014). Coupling between endocytosis and sphingosine kinase I recruitment. Nature cell biology, 16(7), 652-662.
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9

Differentiation of Neural Stem Cells

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Neurospheres were dissociated and cells were seeded at a density of 30 K/cm2 onto #1.5 acid-washed coverslips coated with laminin (10 µg/ml). After 24 h, media was removed and differentiation media was added (same composition as the proliferation media, but without bFGF, EGF, and heparin). Cells were fed every 2 d and fixed after 4 d or fixed after 10 d in differentiation media. For cholesterol rescue experiments, cholesterol–methyl-β-cyclodextrin (#C4951, Lot #SLCB4694; Sigma-Aldrich) at 10 µg/ml (equivalent to 1.3 µM cholesterol) was added to the media at the time of plating and during media changes. Antibodies used for identifying differentiated cell types include astrocytic marker glial fibrillary acidic protein (GFAP), neuronal marker microtubule associated protein (MAP2), and oligodendrocyte marker (O4). GFAP staining of astrocytes is distinguished by a high GFAP signal compared to very low levels found in proliferating NSCs. See Table S3 for antibodies used.
Nourse J.L., Leung V.M., Abuwarda H., Evans E.L., Izquierdo-Ortiz E., Ly A.T., Truong N., Smith S., Bhavsar H., Bertaccini G., Monuki E.S., Panicker M.M, & Pathak M.M. (2022). Piezo1 regulates cholesterol biosynthesis to influence neural stem cell fate during brain development. The Journal of General Physiology, 154(10), e202213084.
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10

Polarization of Bone Marrow-Derived Macrophages

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BMMФ were stimulated either with IL-4 (10 ng/ml, AF-214-14, Peprotech), lipopolysaccharide (100 ng/ml, Sigma) or dexamethasone (100nM, D4902, Sigma) to respectively achieve M(IL-4), M(LPS) or M(GC) polarization state. In some experiments, BMMФ were treated with C75 (Sigma), Cerulenin (Sigma), Etomoxir (Sigma), pan-AKT inhibitor MK-2206 (1 mM, Cayman Chemical), 25-hydroxycholesterol (10 μM, Cayman chemical), N-acetyl cysteine (10mM, Sigma), Diphenyleneiodonium chloride (DPI, 5 μM, Sigma), L-NG-Nitroarginine methyl ester (L-NAME, 1mM, Cayman Chemical), Allopurinol (100 μM, Sigma), hydrogen peroxide (Sigma), SIRT1 activator II (Sigma), EX-527 (Sigma), Compound C (Sigma), AICAR (Abcam), HMGCoA (Sigma), water-soluble cholesterol (Cholesterol–methyl-β-cyclodextrin, Sigma) or Simvastatin (Sigma).
For fatty acid treatment, palmitic or oleic acid (Cayman) were solubilized at 100mM in absolute ethanol at 60°C. Fatty acid were conjugated at the desired concentration using a sonicator water bath into the macrophage culture medium to avoid endotoxin contamination from BSA54 (link).
Bidault G., Virtue S., Petkevicius K., Jolin H.E., Dugourd A., Guénantin A.C., Leggat J., Mahler-Araujo B., Lam B.Y., Ma M.K., Dale M., Carobbio S., Kaser A., Fallon P.G., Saez-Rodriguez J., McKenzie A.N, & Vidal-Puig A. (2021). SREBP1-induced fatty acid synthesis depletes macrophages antioxidant defences to promote their alternative activation. Nature metabolism, 3(9), 1150-1162.
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