Cholesterol assay kit

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Cholesterol Assay Kit is a laboratory diagnostic tool designed to measure the concentration of cholesterol in biological samples. It is a colorimetric assay that provides a quantitative determination of total cholesterol levels.

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8 protocols using cholesterol assay kit

Isolation and Characterization of Oxidized LDL

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Normal human plasma (Innovative Research) was mixed with 12% of OptiPrep density gradient medium (Sigma) at the ratio (v:v) of 1 to 1. The mixture was loaded to the centrifuge tube and placed in a NVT65 rotor (Beckman Coulter), then centrifuged at 60,000 rpm (342,000 g) for 4 hours at 16°C in an Optima L-90K ultracentrifuge (Beckman Coulter) set at slow acceleration and slow deceleration. Samples were fractionated within 1 hour after centrifugation. Fractions were collected from each gradient by downward displacement using a syringe tip piercing the bottom of the tube and pumped out. The fractions were collected into Eppendorf tubes with 1.5 mL per fraction. The density and concentration of each fraction were measured by using a refractometer (ATAGO) and cholesterol assay kit (Invitrogen), respectively. Oxidized-LDL (ox-LDL) was prepared according to a previously reported method.25 (link) CuSO₄ was added to native LDL (n-LDL) with a final concentration of 10 µmol/L. Oxidation was carried out at room temperature over 24 hours until oxidation was complete. The ox-LDL was then placed in ultra-centrifuge tubes (Sigma-Aldrich, Ultra-4, MWCO 30 kDa) and centrifuged at 3,000 rpm for 20 minutes to remove CuSO₄. All of the LDL samples were filtered and stored at 4°C.

Hua J, & Malinski T. (2019). Variable Effects Of LDL Subclasses Of Cholesterol On Endothelial Nitric Oxide/Peroxynitrite Balance – The Risks And Clinical Implications For Cardiovascular Disease. International Journal of Nanomedicine, 14, 8973-8987.

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Cholesterol efflux in macrophages

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Control and CES1KD macrophages were pretreated for 24 h with DMSO vehicle or GW1929/9cRA (1 μM/10 μM) and GSK2033 (1 μM) in culture medium containing 1% FBS. The culture medium was then removed and replaced with fresh medium containing acLDL (50 μg/mL) and 0.2% w/v BSA. After loading the cells with cholesterol for 24 h, the acLDL-containing medium was removed and the cells washed with 1 x PBS. The cells were then overlaid with equilibration medium (containing 0.2% w/v BSA) and incubated for an additional 18 h. The medium and cells were harvested separately, and cells scraped into 0.1 M potassium phosphate (pH 7.4), 0.05 M NaCl, 5 mM cholic acid, 0.1% Triton X-100 and lysed by sonication. Following brief centrifugation to remove cell debris (1,000 x g, 5 min, 4°C), intracellular total cholesterol and free cholesterol levels in the supernatants were assayed using an Invitrogen Cholesterol Assay kit per the manufacturer’s instructions and normalized on cellular DNA content.

Mangum L.C., Hou X., Borazjani A., Lee J.H., Ross M.K, & Crow J.A. (2018). Silencing Carboxylesterase 1 in Human THP-1 Macrophages Perturbs Genes Regulated by PPARγ/RXR and RAR/RXR: Downregulation of CYP27A1-LXRα Signaling. The Biochemical journal, 475(3), 621-642.

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Modulation of Lipid Metabolism Pathways

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Reagents Sorafenib (multikinase inhibitor), T0901317 (LXR pan-agonist), GW3965 (LXR pan-agonist), PF-04217903 (ATP-competitive Met inhibitor), Gefitinib (EGFR-tyrosine kinase inhibitor), MK-2206 (Akt1/2/3 inhibitor), SCH772984 (ERK1/2 inhibitor), and SB202190 (p38 MAPK Inhibitor) were purchased from Selleck Chemicals (Houston, TX, US). Antibodies against LXRα and LXRβ were purchased from Abcam cooperation (Cambridge, UK). All other antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Puromycin and TRIzol reagent were purchased from Thermo Fisher Scientific (Grand Island, NY, USA). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Laboratories (Mashikimachi, kamimashiki gun Kumamoto, JAPAN). PI/RNase Staining Buffer and FITC Annexin V Apoptosis Detection Kit were purchased from BD Biosciences (San Diego, CA, USA). Cholesterol Assay Kit was purchased from Invitrogen (San Diego, CA, USA). BCA Protein Assay Reagent was purchased from Beyotime Biotechnology (Shanghai, China).

Shao W., Zhu W., Lin J., Luo M., Lin Z., Lu L., Jia H., Qin L., Lu M, & Chen J. (2019). Liver X Receptor Agonism Sensitizes a Subset of Hepatocellular Carcinoma to Sorafenib by Dual-Inhibiting MET and EGFR. Neoplasia (New York, N.Y.), 22(1), 1-9.

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Cholesterol and Cholesteryl Esters Quantification

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Cells were washed with PBS twice and collected by scraping and centrifugation at 200 x g for 10 min. The cell pellets were resuspended in Isopropanol/1% Triton X-100 for 1 h at room temperature. After centrifugation at 12,000 rpm (Sorvall Legend Micro 17 centrifuge) for 10 min, the supernatants were transferred into glass tubes and dried under passing nitrogen gas. Cholesterol and cholesteryl esters (CE) measurements were performed following the instruction manual of the cholesterol assay kit (Invitrogen).

Britton B.J., Evans J.G, & Potter J.M. (1998). Does the fly matter? The CRACKPOT study in evidence based trout fishing. BMJ : British Medical Journal, 317(7174), 1678-1680.

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Measuring Cellular Membrane Cholesterol

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The level of plasma membrane (PM) cholesterol was measured by following the previous publication.^{44 (link)} As shown in Figure S5C, 2 x 10⁵ cells were seeded in 60 mm dish in DMEM medium containing 5% FBS. After 24 h, cells were washed with PBS, and then replaced with fresh medium containing 5% LPDS. After 24hr, cells were washed with cold-PBS for 3 times, and 3 times with cold assay buffer (310 mM sucrose, 1 mM MgSO4, 0.5 mM sodium phosphate [pH 7.4]), followed by incubated in the absence or presence of 1 U/ml cholesterol oxidase for 3 min at 25°C, the buffer was then removed and discarded. 2 mL of PBS were used to scape cells, and mixed with 4 mL of chloroform:methanol (2:1) to extract lipid. Finally, the dried lipid was dissolved in 200 μL of 1 x assay buffer from cholesterol measurement kit, and measure the cholesterol level following the instruction manual of the cholesterol assay kit (Invitrogen).

Britton B.J., Evans J.G, & Potter J.M. (1998). Does the fly matter? The CRACKPOT study in evidence based trout fishing. BMJ : British Medical Journal, 317(7174), 1678-1680.

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Cholesterol Dissolution in Plaque

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To observe cholesterol dissolution within plaques after CSNP treatment in the LCA-ligation model, ApoE^−/− mice were injected intravenously with CSNP (200 mg/kg cyclodextrin and 30 mg/kg statin), liposomal statin (30 mg/kg statin), statin (30 mg/kg), cyclodextrin (400 mg/kg), or PBS at 13 days after partial ligation surgery. Twenty-four hours after the injection, the left carotid arteries were collected and put into PBS, respectively. The samples were then incubated at 37 °C in the shaking incubator for 24 h. Dissolved cholesterol in the PBS was measured using a cholesterol assay kit (Catalog Number A12216; Thermo Fisher Scientific) in accordance with the instructions provided by the manufacturer.

Kim H., Kumar S., Kang D.W., Jo H, & Park J.H. (2020). Affinity-Driven Design of Cargo-Switching Nanoparticles to Leverage a Cholesterol-Rich Microenvironment for Atherosclerosis Therapy. ACS nano, 14(6), 6519-6531.

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Biochemical Analysis of Brain Tissue

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Brain tissues were homogenized in RIPA buffer (MilliporeSigma) containing complete protease inhibitors (Roche) and PhosSTOP phosphatase inhibitors (Roche). After centrifugation (100,000g for 1 hour at 4°C), the supernatant was used for biochemical analysis as described (53 (link)). ApoE (53 (link), 55 (link)), collagen IV (LS-F20750, LSBio), MMP-2 (MMP200, R&D Systems), and MMP-9 (NBP2-60095, Novus Biologicals) in brain lysate were measured by ELISA. Brain lysate measurements were normalized to total protein concentrations determined by BCA assay (Thermo Fisher Scientific). Total cholesterol and triglycerides in plasma were measured using a Cholesterol Assay Kit (A12216, Thermo Fisher Scientific) and Triglyceride Assay Kit (ab65336, Abcam), respectively. Some brain samples were subjected to Western blotting using anti–Iba-1 antibody (17198, Cell Signaling Technology) and anti–β-actin antibody (3700, Cell Signaling Technology), followed by quantification through LI-COR Odyssey.

Oue H., Yamazaki Y., Qiao W., Yuanxin C., Ren Y., Kurti A., Shue F., Parsons T.M., Perkerson R.B., Kawatani K., Wang N., Starling S.C., Roy B., Mosneag I.E., Aikawa T., Holm M.L., Liu C.C., Inoue Y., Sullivan P.M., Asmann Y.W., Kim B.Y., Bu G, & Kanekiyo T. (2023). LRP1 in vascular mural cells modulates cerebrovascular integrity and function in the presence of APOE4. JCI Insight, 8(7), e163822.

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High-Fat Diet and LoVo Xenograft

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Twenty 4-week old nude mice were randomly assigned to two groups: the standard chow diet group (n = 10) and the high fat diet (HFD) group (n = 10). Mice in the HFD group were treated with a dietary chow consisting of 60% kcal fat (D12079B) (Research Diets, New Brunswick, USA). The mice were treated with standard chow diet or HFD for 2 weeks, and then LoVo cells (1 × 10 6 for each mouse) were injected into the backside of the five randomly selected mice for the HFD group and the standard chow group. The mice were killed and their weights were measured at week 4, and tumor volumes of each mouse were measured weekly as length × width 2 /2. The blood LDL levels of the mice were assessed following the instruction manual of the cholesterol assay kit (Thermo scientific).

Wang C., Li P., Xuan J., Zhu C., Liu J., Shan L., Du Q., Ren Y, & Ye J. (2017). Cholesterol Enhances Colorectal Cancer Progression via ROS Elevation and MAPK Signaling Pathway Activation. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology, 42(2).

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