Cholesterol/ Cholesteryl Ester Assay Kit - Quantitation

• Biochemical assay

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Biochemical assay - Cholesterol/ Cholesteryl Ester Assay Kit - Quantitation (AB65359)

Diagram showing the principles of the Cholesterol - Cholesterol Ester assay method.

• Biochemical assay

PubMed

Biochemical assay - Cholesterol/ Cholesteryl Ester Assay Kit - Quantitation (AB65359)

Cholesterol Efflux Assay Kit ab196985 and Cholesterol/Cholesteryl ester Assay Kit ab65359 used with mouse bone marrow-derived macrophages.

Wang et al used Cholesterol Efflux Assay Kit ab196985 and Cholesterol/Cholesteryl ester Assay Kit ab65359 to investigate the impact of SULT2B1, the key enzyme of sterol sulfonation, deficiency in Bone marrow-derived macrophages (BMDMs) on cholesterol metabolism in mice.

Intracellular cholesterol content in WT and Sult2b1-/- M2 BMDMs (n = 5 per group). Cholesterol efflux in WT and Sult2b1-/- M2 BMDMs (n = 5 per group). Data are presented as the mean ± SD.

The free-cholesterol content of M2 BMDMs was measured using the cholesterol/cholesteryl ester quantitation assay kit (ab65359; Abcam). In brief, the dried lipids (extracted from 1 Ч 106 M2 BMDMs) were dissolved in 200 µl assay buffer (supplied in the kit), and then incubated with a cholesterol probe/cholesterol enzyme mix solution (supplied in the kit) at 37°C for 1 h protected from light. Finally, the fluorescence was detected by a fluorescent microplate reader (Ex/Em = 535/587 nm). The free-cholesterol content was quantified using a cholesterol standard curve.

The cholesterol efflux was measured using the commercial cholesterol efflux assay kit (cell-based) (ab196985; Abcam). In brief, M2 BMDMs (1 Ч 105) were incubated with a labelling reagent for 1 h and then with the Equilibration Buffer overnight. The cells were treated with the desired cholesterol acceptor (serum) for 6 h in an incubator (37°C, 5% CO2). The fluorescence intensity of the media and cell lysates was measured via a fluorescent microplate reader (Ex/Em = 482/515 nm). The cholesterol efflux was calculated by dividing the fluorescence intensity of the media by the total fluorescence intensity of the cell lysate and media.

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Functional Studies - Cholesterol/ Cholesteryl Ester Assay Kit - Quantitation (AB65359)

Assessment of free and total cholesterol and cholesteryl ester levels useing ab65359. Protein levels of geranylgeranyldiphosphate synthase were measured by ELISA. Absolute amounts of CoQ10 were measured by HPLC as described in Materials and Methods. Cholesterol levels were quantified using a Cholesterol/Cholesteryl Ester Detection Kit from Abcam according to instruction. n = 6, means ±SEM. Two-way ANOVA with Tukey's post-hoc test. * denotes statistical comparison between vehicle control (V) and high dose (H) and # denotes statistical comparison between water and ethanol treated groups. * and **, indicate significant difference at p<0.05, 0.01 and 0.0001, respectively. OWV; obese water vehicle, OWH; obese water high dose, OEV; obese ethanol vehicle, OEH; obese ethanol high dose.

Mailloux RJ, Florian M, Chen Q, Yan J, Petrov I, Coughlan MC, et al., PLoS ONE 9(9): e106832., Fig 3, doi.org/10.1371/journal.pone.0106832

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Functional Studies - Cholesterol/ Cholesteryl Ester Assay Kit - Quantitation (AB65359)

Cholesterol and cholesteryl ester were quantified colourimetrically in biological fluids (dilution range 1 : 10-1 : 100; 50 minutes incubation) and measured in duplicates (+/- SD).

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Functional Studies - Cholesterol/ Cholesteryl Ester Assay Kit - Quantitation (AB65359)

Detection of Cholesterol/Cholesteryl Ester Using Cholesterol Quantitation Kit (ab65359). Cholesterol/Cholesteryl Ester was quantified using the kit by colorimetric (A) and fluorometric (B) methods according to the kit instructions. Background from the control reaction (without cholesterol) has been subtracted from each value. Note : Fluorometric assay is over 10 fold more sensitive than colorimetric assay.

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Functional Studies - Cholesterol/ Cholesteryl Ester Assay Kit - Quantitation (AB65359)

Total cholesterol (free and ester forms) were determined with a cholesterol quantification assay (fluorometric detection) (ab65359) and are shown relative to untreated control cells.

Singh, Natesh and Gerhard F Ecker., International journal of molecular sciences vol. 19,5 1278., Fig 1, doi:10.3390/ijms19051278

• Biochemical assay

PubMed

Biochemical assay - Cholesterol/ Cholesteryl Ester Assay Kit - Quantitation (AB65359)

Cholesterol/ Cholesteryl Ester Assay Kit ab65359 used to measure total cholesterol in liver tissue.

Guo et al. used Cholesterol/ Cholesteryl Ester Assay Kit ab65359 to investigate the role of SIRT1 in cholesterol metabolism.

The effect of a high fat diet (white) versus a high fat diet plus metformin treatment (blue hatching) on wildtype and SIRT1 deficient mice.

The cholesterol content in the liver were measured using a cholesterol quantification colorimetric kit (K603-100, Biovision, CA, United States [Biovision is an Abcam company, this product is now sold as ab65359) according to the manufacturer’s protocols.

• Biochemical assay

PubMed

Biochemical assay - Cholesterol/ Cholesteryl Ester Assay Kit - Quantitation (AB65359)

Triglyceride assay kit ab65336 and cholesterol assay kit ab65359 used with HepG2 cell lysates.

Pozzi et al. used used the triglyceride assay kit and cholesterol assay kit to investigate the effect of antipsychotics on the SREBP pathway and on AMPK activation.

HepG2 cells serum-starved overnight and pre-treated with DMEM low glucose 20% FBS for 3 h, were incubated with 5 μM U18666A, 25 μM risperidone (RIS), ziprasidone (ZIP) or olanzapine (OLA), the SREBP2 positive control lovastatin 25 μM (LOV), and the SREBP1 positive control T0901317 10 μM (T09) for 24 h.

Triglycerides (TG) were extracted and quantified from HepG2 cells treated with the indicated compounds using Abcam ab65336. Briefly, 5 x 10^6 cells were harvested and lysed in 5% v/v NP-40, then samples were repeatedly heated at 98°C to solubilise triglycerides; a sample was taken for BCA protein quantification as normaliser, and the indicated amount of sample was processed with lipase and reaction reagents. Fluorescence emitted by the chromogen dye was quantified on Fluoroskan microplate reader (Ascent FL, Thermo Fisher Scientific) at 544/590 nm. Triglyceride fluorescence was reported as fold increase over levels of untreated cells (one way ANOVA followed by Dunnett's multiple comparison test, n > 3 experiments; *vs. unt cells).

Total cholesterol was extracted from HepG2 cells treated with the indicated compounds by using cholesterol assay kit ab65359. Briefly, 10^6 cells were harvested and lysed in 100 mM NaCl, 10 mM TRIS pH 7.4, 1 mM EGTA, 2 mM MgCl2 and 1% v/v Triton-X100; samples were repeatedly vortexed and chilled on ice to solubilise cholesterol; a sample was taken for BCA protein quantification as normaliser, while the rest was mixed with isopropanol-chloroform 11 : 7 and centrifuged to separate the organic phase containing cholesterol. The organic phase was taken and evaporated, cholesterol was then resuspended and processed with cholesterol esterase and reaction reagents. Fluorescence emitted by the chromogen dye was quantified on Fluoroskan microplate reader at 544/590 nm. Cholesterol fluorescence was reported as fold increase over levels of untreated cells (one way ANOVA followed by Dunnett's multiple comparison test, n > 3 experiments; *vs. unt cells).

• Biochemical assay

PubMed

Biochemical assay - Cholesterol/ Cholesteryl Ester Assay Kit - Quantitation (AB65359)

Alice et al used Cholesterol Efflux Assay Kit ab196985 and Cholesterol/Cholesteryl ester Assay Kit ab65359 with Listeria monocytogenes (Lm) infected human monocyte-derived dendritic cells (Mo-DC) .

Changes in cholesterol metabolism in Lm-infected Mo-DC. (B) Cholesterol content and (C) cholesterol efflux from uninfected or Lm-infected Mo-DC were quantified at 6 hpi as described in “Methods ” section. Results shown are representative of 2 biologically independent experiments. Data represents the mean +/- SEM of each group (n = 3).

Cholesterol was quantified in Lm-infected or uninfected Mo-DC using the Choelsterol/Cholesteryl ester assay kit as described by the manufacturer (#ab65359 Abcam, Cambridge, MA). In general infected cells were processed after 6 h post-infection. Cholesterol efflux was determined using the Cholesterol efflux assay kit (ab196985, Abcam, Cambridge, MA) following manufacturer’s instructions and the recommended positive control.

• Biochemical assay

PubMed

Biochemical assay - Cholesterol/ Cholesteryl Ester Assay Kit - Quantitation (AB65359)

Cholesterol/ Cholesteryl Ester Assay Kit ab65359 used with mouse plasma.

Christensen et al. used Cholesterol/ Cholesteryl Ester Assay Kit ab65359 to investigate the impact of a Western high-fate high sugar diet in human APO3 and APOE4 knock-in mice.

Metabolic profiles in APOE mice after Western diet. Plasma was collected under fasting conditions at the end of the experiment and measured for cholesterol. Data are shown as individual values (open circles) and mean values (+SEM) from n = 7–8 mice per group. "G" indicates a significant main effect of the APOE genotype, "D" indicates a significant main effect of diet, "g" indicates a significant difference from the APOE3 mice on the same diet, "d" indicates a significant difference from animals with the same APOE genotype on the control diet. Significance is p < 0.05.

Plasma cholesterol was measured using a colorimetric cholesterol quantitation assay kit (Abcam, Catalog #ab65359), according to the manufacturer's directions.

• Biochemical assay

PubMed

Biochemical assay - Cholesterol/ Cholesteryl Ester Assay Kit - Quantitation (AB65359)

Cholesterol/ Cholesteryl Ester Assay Kit ab65359 used to measure total cholesterol, free cholesterol, and esterified cholesterol in mouse testicular tissue.

Moutard et al. used Cholesterol/ Cholesteryl Ester Assay Kit ab65359 to investigate testicular tissue development in prepubertal mice.

Intratesticular concentrations of total, free, and esterified cholesterol normalized to tissue mass. Data are presented as means ± SEM with n=4 biological replicates for each group.

Total testicular lipids were extracted from ~10 mg tissue with a Lipid Extraction kit (ab211044; Abcam, Paris, France) according to the manufacturer’s instructions. Dried lipid extracts were reconstituted in 200 µL of assay buffer. Intratesticular levels of total, free, and esterified cholesterol were measured with a colorimetric Cholesterol/Cholesteryl Ester assay kit (ab65359; Abcam) according to the manufacturer’s instructions.

• Biochemical assay

PubMed

Biochemical assay - Cholesterol/ Cholesteryl Ester Assay Kit - Quantitation (AB65359)

Cholesterol/ Cholesteryl Ester Assay Kit ab65359 used to measure intra-cellular cholesterol.

Christensen et al. used Cholesterol/ Cholesteryl Ester Assay Kit ab65359 to investigate the impact of a Western high-fate high sugar diet in human APO3 and APOE4 knock-in mice.

The cholesterol (chol.) level in AML12 cells was reduced (cholesterol depletion; CD) by treatment with lovastatin and mevalonate (50 μM each) in medium containing 10% LPDS (for control samples, 10% FCS), or elevated (cholesterol enrichment; CE) by incubation with cholesterol-MβCD complex (5 mM MβCD, 300 μg/ml cholesterol) in complete growth medium for 16 h, followed by 30 min serum starvation under the same conditions (see "Methods"). The level of free (non-esterified) and total cholesterol was determined using the Abcam cholesterol assay kit ("Methods"). In each experiment, the values obtained in untreated (control) cells were taken as 100%. a Cholesterol depletion. Data are mean ± SEM of four independent experiments. b Cholesterol enrichment. Bars are mean ± SEM of six independent experiments. The results show similar levels of reduction (a) or elevation (b) in total and free cholesterol. c Effect of statin-mediated CD on free cholesterol level at the plasma membrane. After the CD treatment, the membrane cholesterol was extracted by short incubation with a high level of MβCD (30 mM, 30 min, 37 °C), and the free cholesterol level was assayed. Data are mean ± SEM of four independent experiments. d Effect of CE on the free plasma membrane cholesterol. The CE treatment was followed by extraction of the membrane cholesterol by a short exposure to MβCD as described in (c). Data are mean ± SEM of three independent experiments. Asterisks indicate significant differences between the pairs indicated by the brackets. ns not significant. In (a, b), significance was calculated using Student's two-tailed t test. In (c, d), significance was evaluated using one-way ANOVA followed by Bonferroni post hoc test (*p < 0.05, **p < 0.01, ***p < 0.001).

The effects of the CD and CE treatments on the cellular cholesterol content were measured using the Abcam cholesterol assay kit (cat. #ab65359) with or without cholesterol esterase to measure the levels of total and free (membrane-associated) cholesterol, respectively.

• Schematic Diagram

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Schematic Diagram - Cholesterol/ Cholesteryl Ester Assay Kit - Quantitation (AB65359)

Representative image of Cholesterol/ Cholesteryl Ester Assay Kit - Quantitation ab65359

Components shown from left to right :

- OxiRed Probe

- Cholesterol Esterase

- Cholesterol Standard

- Enzyme Mix I

- Assay Buffer 2

Note : The vial labels shown in this image use generic names for illustrative purposes only and may not exactly match the specific component names included in the kit.

Note : Colors of solutions in image may not precisely match the shade of colors in the actual kit.