Gm csf

Approximately 40 mg of brain tissue lysed in RIPA buffer including protease inhibitors were homogenized and centrifuged at 12,000 rpm for 5 min at 4 °C. The concentration of protein was detected by a BCA Protein Quantitative Kit (Dingguo Changsheng Biotechnology, Beijing, China). Equivalent amounts of protein samples (40 ng) were separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes (PVDF). The antibodies used were as follows: APP 1:1000 (Abcam, ab126732, Cambridge, UK), SAA 1:1000 (Abcam, ab199030, Cambridge, UK), CYP27A1 1:1000 (Abcam, ab126785, Cambridge, UK), CYP7B1 1:1000 (ABclonal, A17872, Wuhan, China), CYP46A1 1:2000 (Abcam, ab244241, Cambridge, UK), RORγt 1:2000 (Abcam, ab207082, Cambridge, UK), Foxp3 1:1000 (Abcam, ab215206, Cambridge, UK), IL-17A 1:3000 (Abcam, ab189377, Cambridge, UK), GM-CSF 1:1000 (Proteintech, 17762-1-AP, Chicago, IL, USA), MIP-3α 1:1000 (Abcam, ab106151, Cambridge, UK), IL-10 1:1000 (Abcam, ab189392, Cambridge, UK), IFN-λ2 0.1 µg/mL (R and D, AF4635, Minneapolis, MN, USA). The protein density was measured by Image System Fusion FX (Vilber Lourmat, Paris, France) and GAPDH was used as the reference for standardization.

Human monocyte-derived immature DCs were obtained from peripheral blood mononuclear cells (PBMC) extracted from healthy human ethylene diamine tetraacetic acid (EDTA) anticoagulated whole blood. PBMC were extracted using Ficoll (GE, Boston, MA, United States) and seeded at a density of 5 × 10⁵ cells/mL in 24-well plates. For the induction of cells, human granulocyte-macrophage colony-stimulating factor (GM-CSF, 100 ng/mL, Proteintech) and human interleukin-4 (IL-4, 50 ng/mL, Proteintech) were added to each well. On day 4, the cell culture supernatant of each group was co-cultured with immature DCs. On day 8, positive control groups were treated with 10 μg/mL LPS or 50 ng/mL TNF-α for 24 h.
On day 9, cell surface markers of DCs were evaluated by flow cytometry. DCs were stained on ice for 30 min with FITC-conjugated anti-CD11c and phycoerythrin (PE)-conjugated anti-CD83 (Biolegend, San Diego, CA, United States), and data were acquired and analyzed on a FACSCanto II flow cytometer (BD Biosciences).