3h cholesterol

BV-2 microglia were seeded in DMEM (10% FBS), at a density of 75 K per well of a 12 well plate. The cholesterol efflux assay was adapted from [35 (link)]. When the cells had adhered to the plate (around 1 h), [³H]cholesterol (Perkin Elmer) containing media was added to a final concentration of 0.5 μCi per well for 24 h. [³H]cholesterol-containing media was removed and the cells were washed three times in sterile PBS. Serum-free DMEM was added to the cells, with T0901317 (supplier) at 0–4 μMol/L for 20 h. Media was removed and 0.1 mL was added to 5 mL of Scintillation fluid and considered as “media counts”. The cells were washed three times with PBS and 0.5 mL of double-distilled water was added to the plates, which were then placed in a −80 °C freezer for one hour to help cells detach. After thawing, 0.1 mL of cell solution was added to 5 mL of scintillation fluid and the reads considered as "cell counts". Due to the slight delay in growth rate in the LPL KD cells, the counts were normalized for cell number and protein content. Cholesterol efflux was determined using the following equation (media counts/(media counts + cell counts) × 100) and expressed as a % of total.

J774A.1 cells were cultured in DMEM supplemented with 10% FBS and 1% Penicillin-Streptomycin (10,000 U/mL). Cells were seeded in a 24-well plate at a density of 1 × 10⁵ cells/well and allowed to grow for 2 days. Cells were then labeled with 1 µCi/mL [³H] cholesterol (Perkin Elmer) in DMEM containing 3% fatty acid-free bovine serum albumin (BSA) (Sigma, A8806) and 5 µg/mL ACAT inhibitor Sandoz 58-035 (Sigma, S9318) and incubated overnight. The next day, cells were washed twice with PBS and equilibrated for 24 h in fresh DMEM media containing 0.3% BSA and 5 µg/mL ACAT inhibitor as described above. Cells were then incubated with DMEM containing 0.1% BSA in the presence of indicated micelles at 20 μM for 4 h at 37 °C. At the end of the incubation, the media was collected. The cells were lysed in 0.5 mL of 0.1% SDS and 0.1 N NaOH, and cell lysate was also collected. The [³H] cholesterol content of medium and cells was measured by liquid scintillation counting using Perkin Elmer Tri-Carb 2910TR (Waltham, MA, USA). Cholesterol efflux was presented as a percentage calculated by media counts divided by the sum of media counts and cell counts as described in previous studies [23 (link),26 (link)].

A cholesterol efflux assay using THP-1 cells was carried out as described previously [12 (link)]. Briefly, THP-1 cells (2.5 × 10⁵ cells/well) were differentiated into macrophages via culture in RPMI-1640 medium containing 100 ng/ml of phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich) supplemented with 0.2% bovine serum albumin (BSA) for 2 days. Culture medium was then changed to RPMI-1640 containing acetylated LDLs (acLDLs) (50 μg of protein/ml), T0901317 (1 mmol/l; Enzo Life Sciences), a liver X receptor (LXR) agonist for promoting expression of ABCA1, ³H-cholesterol (1 μCi/ml; PerkinElmer), and BSA (0.2%). In doing so, the THP-1 macrophages were converted to foam cells. After equilibration with RPMI-1640 supplemented with T0901317 (1 mmol/l) and BSA (0.2%) for 18 h, the cells were incubated with BDS or HDL in RPMI-1640 for 4 h. CEC was calculated as the percentage of radioactivity in the medium as per the following formula: {³H-cholesterol in medium/(³H-cholesterol in medium + ³H-cholesterol in cells)} × 100 − the percentage of passive diffusion in the case of no cholesterol acceptor. All samples were assayed in triplicate.

HDL was isolated from L-FoxO1,3,4 mice and littermate controls using sequential density ultracentrifugation as described above. Isolated HDL samples from 2 mice were pooled per sample. The protein concentration in isolated, pooled HDL was measured by bicinchoninic acid (BCA) assay. BM-derived macrophages from WT C57BL/6J mice were prepared and grown in L cell media consisting of DMEM, 10% FBS, 1% penicillin/streptomycin, and 20% conditioned media from L929 cells. Seven days after isolation, the media were changed to DMEM containing 0.2% FFA-free BSA, 100 μg/mL AcLDL, 3 μM TO901317, and 2 μCi/mL ³H-cholesterol (PerkinElmer) to induce foam cell formation. After 24 hours, macrophages were carefully washed and then incubated for 6 hours with DMEM containing 0.2% FFA-free BSA alone, or with the addition of 50 μg human HDL or 50 μg or 10 μg pooled, isolated HDL from L-FoxO1,3,4 mice or control mice. The media were then collected. Cells were lysed in 0.1 M NaOH. ³H was quantified from the media and cell lysates by liquid scintillation counting. Results are expressed as media counts as a percentage of total counts.

Cells were incubated with 0.5 μCi/mL ³H-cholesterol (PerkinElmer) in 12-well plates (1 × 10⁶ cells/well) at 37 °C for 24 h. After washing with phosphate buffered saline (PBS), the cells were treated with indicated doses of Cav for 24 h in a medium with addition of 2% fatty acid-free BSA (FAFA, Sigma) and 2 μM acetyl-coenzyme A acetyltransferase inhibitor. To enhance the cholesterol efflux from cells, human ApoA1 (10 μg/mL) was added and incubated with the cells for another 24 h. Finally, the cells and the medium were collected, and radioactivity was measured [51 (link)]. The percentage of ³H-cholesterol in the media/total ³H-cholesterol content in the cell was calculated. The percentage of cholesterol efflux to ApoA1 by subtracting cholesterol efflux to BSA was presented.