To determine cholesterol efflux, cells were plated at 2 × 104 cells/well in 96 well plates (100 μl/ml) and labeled with fluorescent-labeled cholesterol for 16 h using reagents in Cholesterol Efflux Assay Kit (Sigma-Aldrich) according to the manufacturer’s protocol. In parallel, incubation with GGPP and 22(S)HC was performed for 15 h and 3 h respectively before washing procedure. Cells were treated or infected as required. After 24 h of incubation, supernatants and cell lysates were transferred into black well plates for fluorescence measurement at λex/λem: 482/515 nm in Infinite 200 PRO multimode reader (Tecan, Maennedorf, Switzerland). Each sample was also assayed for protein quantity using Pierce BCA Protein Assay kit (Thermo Fischer Scientific) and measured at an absorbance of 562 nm. Efflux was calculated as percentage of fluorescence intensity of medium divided by total fluorescence intensity of medium and cell lysate (normalized to protein quantity). The values obtained from unstimulated or uninfected cells were subtracted.
Cholesterol efflux assay kit
Manufactured by Merck Group
Sourced in Italy, United States
The Cholesterol Efflux Assay Kit is a laboratory tool designed to measure the efflux of cholesterol from cells. It provides a quantitative assessment of the ability of samples, such as HDL or other agents, to promote the removal of cholesterol from cells.
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6 protocols using cholesterol efflux assay kit
1
Fluorescent Cholesterol Efflux Assay
2
Nanodiscs mimic HDLs in cholesterol efflux
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The ability of nanodiscs to mimic the function of endogenous discoidal HDLs in promoting cholesterol efflux was evaluated, as described above on NHA cells, untreated or treated with Aβ1-42 oligomers (ranging from 1–10 μM) for 24 h. Briefly, untreated NHA cells were incubated with ACM, free apoA-I (20 μg/mL), apoA-I nanodiscs (20 μg/mL apoA- I, 0.05 μmol lipids), or nanodiscs (0.05 μmol lipids). NHA cells treated with Aβ were incubated with ACM or apoA-I nanodiscs (40 μg/mL apoA- I), both at 37 °C for 4 h. Cholesterol efflux was measured using the Cholesterol Efflux Assay kit (Sigma Aldrich, Milano, Italy), following the manufacturer’s instructions. Cell viability, ABCA1, and GFAP levels were also measured, as above described.
3
Intracellular Cholesterol Efflux Assay
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To detect intracellular cholesterol efflux, a cholesterol efflux assay kit was used (Sigma‐Aldrich). Raw 264.7 macrophages were seeded in 96‐well plate and allowed to adhere for 6 h at 37°C with 5% CO2. After washing with FBS‐free RPMI 1640 medium, the labelling mix was added into wells for 1 h at 37°C protected from light according to the instruction. After that labelling mix was aspirated and replaced with the equilibration mix. The plates were then incubated at 37°C overnight protected from light. After the aspiration of equilibration mix, pre‐treated LDL/VLDL‐depleted serum dilution was used as cholesterol acceptors without or with oridonin 10 μM for 8 h. The fluorescence densities of supernatant and cell lysis buffer from samples were measured by Cytation 5 imaging reader (BioTek), based on which the percent of cholesterol efflux was calculated.
4
Cholesterol Efflux Modulation by Amyloid-β
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To evaluate changes in cholesterol efflux in the presence of Aβ1-42, NHA cells were plated in 96-well plate (15,000 cells/cm2) and incubated with different concentrations of oligomers (1 μM and 10 μM) for 24 or 48 h in an unsupplemented ABM medium. Cholesterol efflux was measured using the Cholesterol Efflux Assay kit (Sigma Aldrich, Milano, Italy), following the manufacturer’s instructions, using Astrocytes’ Conditioned Medium (ACM) as a cholesterol acceptor.
ACM was obtained by collecting the astrocyte culture medium after 48 h of growth. ACM was filtered through a 0.22-μm filter, and the presence of apolipoprotein E (apoE), as cholesterol acceptor, was evaluated by SDS-PAGE gel electrophoresis on a 4–12% Bis-Tris-Glycine gel (Thermo Fisher Scientific, Milano, Italy), followed by immunoblotting analysis using anti-apoE antibody (1:100, Santa Cruz Biotechnology). ApoE bands were visualized by an enhanced chemiluminescence system by Amersham Imager 600 (GE Healthcare Srl, Milano, Italy). The percentage of cholesterol efflux in the samples was calculated according to the formula: C = [Fm/(Fm + Fc)] × 100% where Fm is the fluorescence intensity of the supernatants and Fc is the fluorescence intensity of the cell lysate. Each value of Fm and Fc was corrected for white.
ACM was obtained by collecting the astrocyte culture medium after 48 h of growth. ACM was filtered through a 0.22-μm filter, and the presence of apolipoprotein E (apoE), as cholesterol acceptor, was evaluated by SDS-PAGE gel electrophoresis on a 4–12% Bis-Tris-Glycine gel (Thermo Fisher Scientific, Milano, Italy), followed by immunoblotting analysis using anti-apoE antibody (1:100, Santa Cruz Biotechnology). ApoE bands were visualized by an enhanced chemiluminescence system by Amersham Imager 600 (GE Healthcare Srl, Milano, Italy). The percentage of cholesterol efflux in the samples was calculated according to the formula: C = [Fm/(Fm + Fc)] × 100% where Fm is the fluorescence intensity of the supernatants and Fc is the fluorescence intensity of the cell lysate. Each value of Fm and Fc was corrected for white.
5
Cholesterol Efflux Assay in Podocytes
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Cholesterol efflux was determined using a cholesterol efflux assay kit (Sigma-Aldrich, USA). Differentiated human podocytes (1×105 cells/well) were plated in a 96-well plate. After incubation for 2 h at 37 °C with 5% CO2, the cells were washed with RPMI 1640 medium (without FBS). Then, the appropriate reaction mix was added to label the cells. After being washed with RPMI 1640 medium (without FBS), the cells were incubated for 4 h with RPMI 1640 medium containing 10% FBS at 37 °C with 5% CO2. After incubation, the supernatant was kept, and the cell monolayer in each well was solubilized with 100 μL cell lysis buffer. Finally, the fluorescence (λex=482 nm /λex=515 nm) of the supernatant and the cell lysate was measured by a fluorescence microplate reader.
6
Cholesterol Efflux Assay in Foam Cells
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Cholesterol Efflux Assay Kit (MAK192, Sigma Aldrich, USA) was used to determine whether the samples could activate and aid cholesterol efflux process in macrophage-derived foam cells following the manufacturer's instructions. Infinite® 200 Microplate Reader was used to measure fluorescence intensity (λex = 482/ λem = 515 nm) of the collected cells supernatants.
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