Analyzing Cell-Specific LXR Regulation in Neuroinflammation

Recombinant hCMEC/D3 cell lines (1 × 10⁶ cells/ml) expressing either LXRα shRNA, LXRβ shRNA, or non-targeting shRNA were seeded in 24-well plates in growth medium. Upon confluency, cells were treated with DMSO (VWR, Leuven, Belgium) or with 5 ng/ml TNFα and 5 ng/ml IFNγ (Peprotech, London, UK) for 24 h. EAE animals were sacrificed on day 23 post-adoptive transfer or day 36 post-immunization. Spinal cords were isolated and snap frozen in liquid nitrogen. Total RNA from cultures and tissues was extracted using Qiazol (Qiagen, Venlo, The Netherlands) and the RNeasy mini kit (Qiagen), according to the manufacturer's instructions. RNA concentration and purity were determined with a NanoDrop spectrophotometer (Isogen Life Science, De Meern, The Netherlands). cDNA was synthesized using qScriptTM cDNA SuperMix (Quanta Biosciences, VWR), following manufacturer's guidelines. qRT-PCR was carried out using SYBR green master mix (Applied Biosystems, Waltham, MA) and a Step One Plus detection system (Applied Biosystems). Primers used for qRT-PCR are shown in Table S1. Relative quantitation of gene expression was accomplished using the comparative Ct method. Data were normalized to the most stable reference genes, as previously described (19 (link)).