Oxidized ldl

Cholesterol efflux assays were carried out essentially as described [37 (link)]. Thioglycollate-elicited peritoneal macrophages were seeded in 24-well plates at a density of 3 x 10⁵ cells/well overnight in Macrophage Growth Medium (MGM; DMEM supplemented with 10% FBS and 10% L-929 conditioned medium). Non-adherent cells were removed, and adherent cells were washed twice with PBS and cultured in MGM. The cells were incubated with 50 μg/mL oxidized LDL (Alfa Aesar) and 1 μCi/mL [1,2-³H(N)]-cholesterol (PerkinElmer) in MGM for 24 hours at 37°C. Following incubation, the cells were washed twice with PBS, incubated in low serum growth medium (DMEM supplemented with 1% FBS, penicillin, and streptomycin), and treated with or without 8-Br-cyclic AMP (Sigma) for 16–18 hours. To induce efflux, cells were washed twice with PBS and incubated in DMEM containing 50 μg/mL HDL (Intracel). At the indicated time points, the medium was collected and centrifuged at maximum speed for 10 minutes and cells were harvested by addition of NP-40 lysis buffer. Radioactivity in the medium and cell lysate was quantified by liquid scintillation counting, and percent cholesterol efflux was calculated as follows: (cpm_Media/(cpm_Media + cpm_Cell)) x 100 after subtracting background efflux (efflux in the absence of HDL).

Oxidized-LDL was purchased from Alfa Aesar (Haverhill, MA; Cat No. J65591). Cells were treated at concentration of 10uM for 6 hours with and without α-NF at 10nM (Sigma Aldrich, St. Louis, MO; Cat No. N5757). The changes in downstream genes were confirmed using RT-qPCR.

Calvaria cells were isolated from either C57BL/6J andScarb1 KO or Osx1-Cre and Osx1-Cre;Scarb1^fl/fl littermate neonatal pups mice by sequential digestion with collagenase type 2 (Worthington, Columbus OH, USA, cat. CLS-2, lot 47E17554B) as previously described [27 (link)]. The cells were cultured in α-MEM medium (Invitrogen, Carlsbad, CA, USA, cat. 11900–0.24) containing 10% Premium Select fetal bovine serum (FBS) (Atlanta Biologicals, Flowery Branch, GA, USA), 1% penicillin/streptomycin/glutamine (PSG) and L-Ascorbic Acid Phosphate (Wako, Richmond, VA, USA cat. 013–12061) for twenty-one days. Gene expression was quantified by PCR as indicated below. Bone marrow cells were obtained by flushing the femoral diaphysis (after removing the proximal and distal ends) and culturing with α-MEM medium containing 10% Premium FBS, 1% PSG and 1 mM L-Ascorbic Acid Phosphate up to 80% confluence. Proliferation was measured by BrdU incorporation with the Cell Proliferation ELISA kit from Roche Diagnostics (Roche Diagnostics, Indianapolis, IN, USA) following manufacturer instructions. Triplicate cultures were analyzed for all assays. Oxidized LDL was obtained from Alfa Aesar (Alpha Aesar, Haverhill, MA, USA).