Cholesterol sulfate

Mouse myoblast C2C12 cells (CRL‐1772, ATCC, Manassas, VA, USA) were grown in Dulbecco's modified Eagle's medium (DMEM) (Hyclone, Logan, UT, USA) containing 10% foetal bovine serum (FBS). To differentiate confluent C2C12 cells to myotubes, cells were cultured in DMEM containing 2% horse serum (Gibco™ 16050‐130, Thermo Fisher Scientific, Waltham, MA, USA). To prepare the bovine serum albumin (BSA) (A8806, Sigma‐Aldrich)‐conjugated fatty acids, the media containing 0.5% BSA and 0.1‐mM palmitic acid (PA) were incubated at 37°C for 1 h. Cholesterol sulfate (C9523, Sigma‐Aldrich), JC1‐40 or PA (P5585, Sigma‐Aldrich) conjugated with BSA was added to the differentiation medium at Day 2 after cell seeding. Synthesis and preparation of JC1‐40 have been described.
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Methanol and water, LC/MS grade, were from MercK (Darmstadt, Germany). [25,26,26,26,27,27,27-D7]-cholesterol sulfate standard (CHOS-d) and cholesterol sulfate were purchased from Sigma-Aldrich (Milan, Italy). Reference sterol sulfates were synthesized in-house by chemical derivatization of sterols (24-methylene cholesterol, desmosterol, campesterol, brassicasterol, dihydrobrassicasterol, stigmasterol, fucosterol, and β-sitosterol) that were purchased from Sigma-Aldrich (Milan, Italy). HPLC for synthetic StS standard purification were performed on a JASCO system (PU-2089 Plus quaternary gradient pump equipped with a MD-2018 Plus photodiode array detector and Sedex 85 high-sensitivity LT-ELS detector). UPLC–MS analysis was performed on Q-Exactive hybrid quadrupole-orbitrap mass spectrometer (Thermo Scientific, Waltham, MA, USA) equipped with an Infinity 1290 UHPLC System (Agilent Technologies, Santa Clara, CA, USA).

Ceramide (type III and VI) was kindly supplied by Evonik Industries (Evonik Nutrition & Care GmbH, Essen, Germany). Cholesterol and caffeine were purchased from Hungaropharma Plc. (Budapest, Hungary). Cholesterol sulfate and EDTA were obtained from Sigma-Aldrich (Saint Louis, Missouri, USA). Palmitic acid was supplied by Mosselman s.a. (Ghlin, Belgium). Kolliphor RH40 was obtained from BASF SE (Ludwigshafen, Germany). Transcutol was a kind gift of Azelis Hungary Ltd. (Budapest, Hungary). Organic solvents (chloroform and methanol), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and sodium chloride (purity ≥ 99.5%) were from Carl Roth GmbH & Co. KG (Karlsruhe, Germany). HPTLC plates (20 × 10 cm silica-gel 60 WRF254s) were delivered by Merck (Darmstadt, Germany). Mobile-phase components, solvents for lipids and o-phosphoric acid 85% were purchased from Carl Roth GmbH (Karlsruhe, Germany) and were of HPLC or p.a. quality. HPLC grade water was prepared from Milli-Q water purification system (Merck Type 1, Millipore, Milford, MA, USA) and methanol (gradient grade, suitable for HPLC/UHPLC measurements) was purchased from VWR Scientific (Seattle, WA, USA).

Aβ25–35 and cholesterol sulfate were obtained from Sigma-Aldrich (St. Louis, MO, USA). AA, 9-CRA, Glu Asp, GABA, Gly, Ach, 5-HT, DA, and NE were purchased from Aladdin (Shanghai, China). LA was purchased from Fluka (Muskegon, MI, USA). Leucine enkephalin and sodium formate were obtained from Waters (Milford, MA, USA). Acetonitrile, methyl alcohol, and formic acid (HPLC-grade) were obtained from Tedia Company, INC. (Cincinnati, OH, USA). Ethanol absolute was purchased from Beijing Chemical (Beijing, China). Ethyl acetate was purchased from Tian in Fuyu Fine Chemical Co., Ltd (Tianjing, China). Ultrapure water was obtained using Milli-Q water purification system (Milford, MA, USA). Schisandra chinensis was purchased from Kaida Company (Baoding, Hebei, China), and authenticated by researcher Fengrui Song, Center for Mass Spectrometry in Changchun. AB-8 macroreticular adsorption resin was purchased from Amicogen Biopharm Co. Ltd (Jining, Shandong, China). The assay kits for β-secretase, γ-secretase, GSK-3β, AChE, TNF-α, PGE2, GSH, HMOX1, SOD, NOS, NO, MDA, and CAT were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China).

Primary bone marrow monocytes from C57BL/6 or Sult2b1^-/- mice were first treated with red blood cell lysis buffer (Cat^# 00-4300-54, eBioscience, CA, USA). Then, the cells were plated in 24-well plates at 37 °C in a 5% CO2 incubator. After 12 h, cell media was exchanged with 10 ng/mL Macrophage colony stimulation factor (M-CSF) recombinant mouse protein (Cat^# PMC2044, Thermo Fisher Scientific, Waltham, MA, USA) for 6 days, which differentiated peripheral monocytes into the neutral state macrophages, M (M-CSF). M (M-CSF) can be further polarized to the pro-inflammatory macrophages, M (LPS) using 1 mg/mL lipopolysaccharides (Cat^# L2880, LPS; Sigma-Aldrich, St. Louis, MI, USA), or anti-inflammatory macrophages, M(IL-4) using 2 mg/mL recombinant murine IL-4 (Cat^# 214-14, PeproTech Sciences, Inc., Ontario, Canada). Cholesterol sulfate (Cat^# 700016P-25MG, Sigma, USA) were added to the cell culture at the indicated time points. For oxygen-glucose deprivation (OGD) model, BMDMs were transferred to deoxygenated, glucose-free DMEM cell culture media. Then, cells were placed in an oxygen deprivation box and incubated with 1% O₂, 5% CO₂, and 95% N₂ at 37 °C for 4-6 h to establish the OGD injury model. Following OGD, the cells were transferred to normal cell culture media and cultured with 5% CO₂ at 37 °C for overnight for the re-oxygen and re-glucose treatment.