Cholesterol efflux fluorometric assay kit

Manufactured by Abcam

Sourced in United States

The Cholesterol Efflux Fluorometric Assay Kit is a laboratory tool designed to measure the efflux of cholesterol from cells. It provides a quantitative assessment of cholesterol transport out of cells.

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Lab products found in correlation

T0901317, by Merck Group (1 mentions) Gw3965, by Merck Group (1 mentions) 25 hydroxycholesterol, by Merck Group (1 mentions) K582 100, by Abcam (1 mentions) H1015, by Merck Group (1 mentions) Mbs173147, by MyBioSource (1 mentions) Spectramax gemini em microplate reader, by Molecular Devices (1 mentions) Apoa 1, by Merck Group (1 mentions) Hdl and ldl vldl quantification colorimetric fluorometric kit, by Abcam (1 mentions) Amplex red cholesterol assay kit, by Thermo Fisher Scientific (1 mentions) Cholesterol extraction kit, by Merck Group (1 mentions)

Spelling variants of this product

Cholesterol Assay Kit (97 citations) Fluorometric assay kit (63 citations) Cholesterol Efflux Assay Kit (13 citations) Cholesterol (6 citations) Fluorometric assays (3 citations) Cholesterol efflux assay (2 citations)

12 protocols using cholesterol efflux fluorometric assay kit

Lipid Accumulation and Efflux Measurement

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Oil red O and Dil ox-LDL staining were used to detect lipid accumulation in the cells. For the oil red O staining, at the indicated post-PDT time points, the cells were stained with freshly diluted 0.5% oil red O solution for 10 min at 37 °C. After washing twice with PBS, the cells were visualized and photographed under a fluorescence microscope (IX-71; Olympus). For the Dil ox-LDL staining, THP-1 macrophages were incubated with 30 μg/ml of Dil ox-LDL diluted with RPMI 1640 medium for 6 h at 37 °C in the dark. After washing twice with PBS, the cells were incubated with 5 μg/ml of 4',6-diamidino-2-phenylindole (DAPI) for 15 min in the dark. Then, the cells were washed with PBS, visualized and photographed under a fluorescence microscope.
The cholesterol efflux in the cells was quantified using the Cholesterol Efflux Fluorometric Assay Kit (Biovision, San Francisco, USA) according to the manufacturer’s protocol. The cholesterol efflux of the treatments was calculated by dividing the fluorescence intensity of the medium by the total fluorescence intensity of the cell lysate and the medium.

Han X.B., Li H.X., Jiang Y.Q., Wang H., Li X.S., Kou J.Y., Zheng Y.H., Liu Z.N., Li H., Li J., Dou D., Wang Y., Tian Y, & Yang L.M. (2017). Upconversion nanoparticle-mediated photodynamic therapy induces autophagy and cholesterol efflux of macrophage-derived foam cells via ROS generation. Cell Death & Disease, 8(6), e2864-.

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Cholesterol Efflux Assay with Astrocytes and Microglia

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Isogenic APOE astrocytes and microglia were seeded at 90% confluency on Matrigel-coated 96 well plates and maintained in growth media (2% serum for astrocytes, serum free for microglia). After 24 h, cells were washed with serum free media, loaded with fluorescently labeled cholesterol (Biovision, K582–100) that performs equivalently to radiolabeled cholesterol for 1 h and then equilibrated o/n in the incubator with light protection. Cells were treated with either cholesterol acceptors or methyl-β-cyclodextrin (MBCD) according to the manufacturers protocol using a cholesterol efflux fluorometric assay kit (Biovision, K582–100) and incubated an additional 4 – 6 h in the incubator with light protection. For LXR agonist treatments, cells were treated with 1x DMSO, 100nM GW3965 (Sigma-Aldrich, G6295), 8µM T0901317 (Sigma-Aldrich, T2320), 4 µM 25-hydroxycholesterol (Sigma-Aldrich, H1015) for 24 h with or without cholesterol acceptors. Further supernatants were transferred to flat-bottom 96 well plates, followed by fluorescent measurement at Ex/Em 485/523 nm. The adherent cells were solubilized by lysis buffer, and the lysates transferred to another 96 well plate where the fluorescence was again measured at the same wavelength. The fraction of total cholesterol was calculated by measuring RFU of supernatant normalized by the total RFU from cell lysate and supernatant.

TCW J., Qian L., Pipalia N.H., Chao M.J., Liang S.A., Shi Y., Jain B.R., Bertelsen S.E., Kapoor M., Marcora E., Sikora E., Andrews E.J., Martini A.C., Karch C.M., Head E., Holtzman D.M., Zhang B., Wang M., Maxfield F.R., Poon W.W, & Goate A.M. (2022). Cholesterol and matrisome pathways dysregulated in astrocytes and microglia. Cell, 185(13), 2213-2233.e25.

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Quantifying Macrophage Cholesterol Efflux

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The capacity of macrophage cholesterol efflux was measured using Cholesterol Efflux Fluorometric Assay Kit (BioVision, Inc., USA) by following the manufacturer’s instructions. After treatment, foam cells in 96-well plate (black plate) were incubated with fluorescence-labeled cholesterol for 16 h at 37 °C. Then, cells were treated with cholesterol acceptors high-density lipoprotein (50 μg/well). Six hours later. After 6 h treatment, the supernatant and cell monolayer solubilized with cell lysis buffer were transferred to a 96-well plate (white plate) and measured at 482/515 nm. The cholesterol efflux is calculated as follows:

Cholesterol efflux %= Fluorescence intensity of the media / (Fluorescence intensity of the cell lysate + media) \times 100 .

Cao Z., Zhang T., Sun X., Liu M., Shen Z., Li B., Zhao X., Jin H., Zhang Z, & Tian Y. (2019). Membrane-permeabilized sonodynamic therapy enhances drug delivery into macrophages. PLoS ONE, 14(6), e0217511.

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Cholesterol Efflux Assay for Stably Transfected Cells

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The stably transfected cells (2 × 10⁵ cells) or HCT116 cells were treated with BSA, APOA-I, AIBP or the AIBP + APOA-I combination for 6 h at 37 °C in 5% LPDS and DMEM or EBM. Then, cholesterol efflux assays were performed using the Cholesterol Efflux Fluorometric assay kit (Biovision, USA) according to the manufacturer’s instructions. The following equation was used to calculate the cholesterol efflux percentage: % Cholesterol efflux = (Fluorescence intensity of the media) / (Fluorescence intensity of the cell lysate + media) × 100.

Zhang T., Wang Q., Wang Y., Wang J., Su Y., Wang F, & Wang G. (2019). AIBP and APOA-I synergistically inhibit intestinal tumor growth and metastasis by promoting cholesterol efflux. Journal of Translational Medicine, 17, 161.

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Macrophage Cholesterol Efflux Assay

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The capacity of macrophage degenerating cholesterol was measured using Cholesterol Efflux Fluorometric Assay Kit (BioVision, Inc., USA) according to the manufacturer's instructions. Briefly, macrophages in 96-well plate (black plate) were firstly incubated with fluorescence-labeled cholesterol for 16 h at 37 °C. After loading with HY, macrophages were treated with cholesterol acceptors high density lipids (HDL; 50 μg/well) alone or for SDT. After different treatments, the supernatant and cell monolayer solubilized by cell lysis buffer were transferred to a 96-well plate (white plate) and measured at 482/515 nm. The cholesterol efflux is calculated as follows: Cholesterol efflux%=Fluorescence intensity of the media/(Fluorescence intensity of the cell lysate+media) × 100.

Li X., Zhang X., Zheng L., Kou J., Zhong Z., Jiang Y., Wang W., Dong Z., Liu Z., Han X., Li J., Tian Y., Zhao Y, & Yang L. (2016). Hypericin-mediated sonodynamic therapy induces autophagy and decreases lipids in THP-1 macrophage by promoting ROS-dependent nuclear translocation of TFEB. Cell Death & Disease, 7(12), e2527-.

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Cholesterol Efflux Quantification Assay

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The total cholesterol efflux in culture medium and cell lysate was measured using a Cholesterol Efflux Fluorometric Assay Kit (BioVision, San Francisco, CA USA) according to the manufacturer’s protocol [24 (link)]. The fluorescence-labeled cholesterol was washed with 200 μL of phenol red-free, serum-free RPMI medium and then incubated in serum-free RPMI medium containing cholesterol acceptor ApoA 1 (50 μg/well) for 16 h at 37 °C. The total fluorescence intensity at 48 h post-transfection or treatment was detected at excitation and emission wavelengths of 482 nm and 515 nm, respectively. Cholesterol efflux was calculated as: 100% x media fluorescence intensity/total fluorescence intensity.

Xie Q., Peng J., Guo Y, & Li F. (2021). MicroRNA-33-5p inhibits cholesterol efflux in vascular endothelial cells by regulating citrate synthase and ATP-binding cassette transporter A1. BMC Cardiovascular Disorders, 21, 433.

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Cholesterol Efflux Fluorometric Assay

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Cholesterol efflux was determined using the cholesterol efflux fluorometric Assay Kit (Cat. K582100; BioVision, Milpitas, CA, USA). Briefly, macrophages were plated on 96-well plates (1X10⁵ /well) and labeled with fluorescently-labeled cholesterol analogue for 1 h. After equilibration in culture for 16 h, cells were incubated without (negative control) or with 10 mM methyl-β cyclodextrin (positive control) or 50 μg/ml human HDL(MBS173147, MyBioSource, San Diego, CA, USA) as indicated in phenol red-free, serum-free RPMI medium for 6 h. The culture medium and cell lysate were separately collected and the fluorescence intensity (RFU) was determined using a microplate reader (SpectraMax Gemini EM Microplate Reader, Molecular Devices, San Jose, CA, USA). The percentage of cholesterol efflux = [RFU of medium/(RFU of cell lysate + RFU of medium)] × 100.

Hsu Y.W., Hsu F.F., Chiang M.T., Tsai D.L., Li F.A., Angata T., Crocker P.R, & Chau L.Y. (2021). Siglec-E retards atherosclerosis by inhibiting CD36-mediated foam cell formation. Journal of Biomedical Science, 28, 5.

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Fluorometric Cholesterol Efflux Assay

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Cholesterol efflux was determined using a cholesterol efflux fluorometric assay kit (BioVision) according to the manufacturer's protocol. Mature adipocytes were incubated overnight with labeling reagent. Then the labeling reagent was removed and the cellular cholesterol efflux to 20 μg/ml of free ApoA-1 (Sigma) or 50 μg/ml of HDL phospholipid (Sigma) was assayed in SILAC advanced medium (Gibco) for 5 h. Cellular and medium fluorescence were measured at Ex/Em = 485/523 nm.

Barilla S., Liang N., Mileti E., Ballaire R., Lhomme M., Ponnaiah M., Lemoine S., Soprani A., Gautier J.F., Amri E.Z., Le Goff W., Venteclef N, & Treuter E. (2020). Loss of G protein pathway suppressor 2 in human adipocytes triggers lipid remodeling by upregulating ATP binding cassette subfamily G member 1. Molecular Metabolism, 42, 101066.

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Cholesterol Efflux Fluorometric Assay

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Cholesterol efflux were detected by Cholesterol Efflux Fluorometric Assay Kit (Biovision, Milpitas) according to the manufacturer's instruction. Briefly, VSMCs were cultured in 96 well plates and stimulated as above. After labelling in a 37°C incubator containing 5% CO2 for 16 hours, cells were treated with 50 μg/mL apoA1 as cholesterol acceptors in DMEM media for 6 hours. At the end of incubation, transfer supernatant to a new 96 well plate and measure the fluorescence (Ex/Em=482/515 nm). Add 100 μL of Cell Lysis Buffer and shaking on a plate shaker for 30 minutes at room temperature to solubilize the cells. Pipette up and down to dissolve any cell debris. Measure the fluorescence (Ex/Em=482/515 nm). % Cholesterol Efflux=Fluorescence intensity of the media/(Fluorescence intensity of the cell lysate+media).

Wang R., Wu W., Li W., Huang S., Li Z., Liu R., Shan Z., Zhang C., Li W, & Wang S. (2018). Activation of NLRP3 Inflammasome Promotes Foam Cell Formation in Vascular Smooth Muscle Cells and Atherogenesis Via HMGB1. Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease, 7(19), e008596.

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Cholesterol Efflux Fluorometric Assay

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The effect of BBR-SDT on cholesterol efflux was measured by using a Cholesterol Efflux Fluorometric assay kit (BioVision, CA, USA) according to the manufacturer's protocol.^{65 (link)} Briefly, 50 μl of labeling reagent and 50 μl of equilibration buffer containing reagent A and B were mixed and added to each well (100 μl of mix/well). THP-1 macrophages were then added to each well and were incubated overnight. After 16 h, the cells were washed and the indicated doses of berberine were added. After 4 h, the cells were washed and incubated with HDL (50 μg/well, Yiyuan Biotechnologies) as cholesterol acceptors, and then were exposed to ultrasound. Six hours later, the supernatants were transferred to a white 96-well plate, the cells were solubilized with cell lysis buffer (100 μl), shaken for 30 min, and fluorescence was measured (Ex/Em=482/515 nm). Cholesterol efflux was calculated by dividing the fluorescence intensity of the media by the total fluorescence intensity of the cell lysate of the same treatment + media.

Kou J.Y., Li Y., Zhong Z.Y., Jiang Y.Q., Li X.S., Han X.B., Liu Z.N., Tian Y, & Yang L.M. (2017). Berberine-sonodynamic therapy induces autophagy and lipid unloading in macrophage. Cell Death & Disease, 8(1), e2558-.

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