Cholesterol Efflux Assay Kit (Cell-based)

• Biochemical assay

Lab

Biochemical assay - Cholesterol Efflux Assay Kit (Cell-based) (AB196985)

Diagram showing the principles of the Cholesterol Efflux assay method.

• FuncS

Supplier Data

Functional Studies - Cholesterol Efflux Assay Kit (Cell-based) (AB196985)

Percentage (%) Labeled Cholesterol Efflux. J774.1 cells were labeled with the fluorescent cholesterol Labeling Media and treated with various cholesterol acceptors such as 2% LDL/VLDL-depleted Human Serum (2 µl/well), purified human HDL (25 µg/ml, 2.5 µg/well) or Positive Control reagent. Cholesterol efflux is expressed as percentage of fluorescently-labeled cholesterol transferred from labeled adherent macrophages to designated cholesterol acceptor present in supernatant over a 4 hour time period, with no treatment control (efflux to medium containing no acceptor) percentage subtracted (mean ± SD of N = 5-6 wells).

• Biochemical assay

PubMed

Biochemical assay - Cholesterol Efflux Assay Kit (Cell-based) (AB196985)

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 Efflux Assay Kit (Cell-based) (AB196985)

Cholesterol Efflux Assay Kit ab196985 used with THP-1 macrophages.

Nyandwi et al used Cholesterol Efflux Assay Kit ab196985 to investigate the effect of Rosmarinic acid (RA) on macrophage cholesterol efflux.

THP-1 macrophages were labeled with cholesterol and equilibrated for 24 h in the presence or absence of RA (1, 10, 50 and 100 µM). Cholesterol efflux was evaluated in THP-1 macrophages after 6 h of incubation with ApoA1 (C) or HDL (D) as described in the Methods section. The data are presented as the mean ± SD of five independent experiments.

Macrophage-specific cholesterol efflux capacity was measured using a commercially available Cholesterol Efflux Assay Kit (Abcam, ab196985, Cambridge, MA, USA). Briefly, THP-1-derived macrophages (1 Ч 105) were labeled with a labeling reagent which includes a fluorescent labelled cholesterol and equilibrated for 24 h in a humidified incubator at 37 °C and 5% CO2. Then, the media was removed, and cells were washed with fresh media and treated with RA (1, 10, 50 and 100 µM) for 24 h. Cells were added with HDL or ApoA1 cholesterol acceptors and incubated for 6 h more. Thereafter, the cholesterol contents in media and cell lysis were measured and cholesterol efflux to HDL or ApoA1 was calculated as follows :

% cholesterol efflux

=100*

fluorescence intensity of medium /

fluorescence intensity of cell lysate+medium

• Biochemical assay

PubMed

Biochemical assay - Cholesterol Efflux Assay Kit (Cell-based) (AB196985)

Cholesterol Efflux Assay Kit ab196985 used with peritoneal macrophage cell cultures.

Hong et al used Cholesterol Efflux Assay Kit ab196985 to investigate the potential mechanism of lncRNA AI662270 on macrophage cholesterol transport in atherosclerosis.

To assess the physiological significance of Abca1 regulation by AI662270, we performed cholesterol efflux assays in peritoneal macrophages. Peritoneal macrophages were transfected with AI662270-P or siAI662270 for 48 h, and treated with either HDL (50 µg/mL) or apolipoprotein A-I (ApoA-I, 25 µg/mL) for 6 h. As shown in Fig. A, the macrophage-specific cholesterol efflux to lipid-poor HDL or ApoA-I capacity was significantly decreased by forced expression of AI662270 in vitro. In contrast, knockdown of AI662270 enhanced cholesterol efflux to lipid-poor HDL or ApoA-I (B).

AI662270 initiates cholesterol efflux and promotes foam cell formation. A, B Cholesterol efflux assay kit was used to measure cholesterol efflux to HDL or ApoA-I in peritoneal macrophages isolated from + AI662270-P (A) or + siAI662270 (B) (n = 5).

Cholesterol efflux was measured using a cholesterol efflux assay kit (ab196985; Abcam). Peritoneal macrophages were incubated with or without the plasmid vector AI662270-P) and AI662270 specific siRNA (siAI662270) as required. The cells were treated with either HDL (50 µg/mL) or ApoA-I (10 µg/mL). The percentage of cholesterol efflux was that the value assessed with fluorescence intensity of media was divided by the value calculated with fluorescence intensity of cell lysate plus fluorescence intensity of media × 100%.

• Biochemical assay

PubMed

Biochemical assay - Cholesterol Efflux Assay Kit (Cell-based) (AB196985)

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.