H1015

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.