Methylumbelliferone

R(+)-NAF (>99.5% ee) and S(-)-NAF (>99.5% ee) (Figure 1) were synthesized according to previous methods (Shivani et al., 2007 (link)). The internal standard (IS) Z10 (CN201110148322.7) was obtained from our laboratory. HPLC-grade methanol, acetonitrile, and acetic acid were obtained from Merck (Darmstadt, Germany). Uridine diphosphate glucuronic acid (UDPGA), alamethicin, Tris, magnesium chloride, methylum-belliferone (MU), 4-methylumbelliferone (4-MU), 4-methylum-belliferyl-β-D-glucuronide hydrate (4-MU-G), propofol, propofol glucuronide, zidovudine, zidovudine glucuronide, fluconazole, and niflumic acid, trifluoperazine dihydrochloride (TFP), and TFP-glucuronide (TFP-G) were purchased from Sigma–Aldrich (St. Louis, MO, United States). Recombinant human UGTs (UGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15, and 2B17) and the commercially available microsomes from human liver (HLMs), intestine (HIMs), and kidney (HKMs), as well as those from rat liver (RLMs), intestine (RIMs), and kidney (RKMs), were purchased from BD Gentest (Woburn, MA, United States). Protein contents of the microsomes were used according to the manufacturers’ instructions. Ultrapure water was used in all experiments (Millipore, Billerica, MA, United States).

LN LECs were seeded sparsely in black, clear-bottom plates (Corning Incorporated, Corning, NY, USA) and left to attach for 4 h. Full medium was replaced with starvation medium (0.5% FBS, 1% penicillin-streptomycin, 1% L-glutamine in αMEM) and the cells were incubated at 37 °C overnight. Then, the medium was replaced by either fresh starvation or full medium and LECs were treated with 10 µg/mL blocking antibody against CD200, tenascin or BST2 or with corresponding isotype controls (Supplementary Table S1) for 48 h. Then, the medium was removed, 0.1 mg/mL methylumbelliferone (Sigma-Aldrich) was added to the cells for 1 h at 37 °C, and the fluorescence was measured using a plate reader (SpectraMAX GEMINI EM, Molecular Devices, San Jose, CA, USA) with excitation 355 nm and emission 469 nm.

Lead nitrate (Pb(NO₃)₂), sodium lauryl sulfate, creatinine anhydrous, propionic acid, lithium d-, and l-lactate, acetylacetone, 37% formaldehyde, cimetidine, indoleacetic acid, methylumbelliferone, and methylumbelliferyl N-acetyl-β-d-glucosaminide were purchased from Sigma-Aldrich Fine Chemicals Inc. (MO, USA). Formic acid, HPLC grade acetonitrile (ACN), ethanol and methanol (MeOH) were purchased from Merck (Darmstadt, Germany). Hippuric acid, 2,2′-dipyridyl disulfate (DPDS), triphenylphosphine (TPP) and 4-nitro-7-piperazino-2,1,3-benzoxadiazole (NBD-PZ) were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). 5-Aminolevulinic acid hydrochloride and guanidinoacetic acid were purchased from Alfa Aesar (MA, USA). Sulbactam was purchased from Cayman Chemical (Michigan, USA). Acetic acid was purchased from Hayashi Pure Chemical Ind.,Ltd. (Osaka, Japan). Trifluoroacetic acid (TFA) was purchased from Riedel-de Haën (Seelze, Germany). 3-(Trimethylsilyl)propionic-2,2,3,3-d₄ acid sodium salt (TSP) was purchased from Cambridge Isotope Laboratories (MA, USA). 1,1-Dimethylbiguanide hydrochloride (metformin) was purchased from Santa Cruz Biotechnology, Inc. (Texas, USA). Protein Assay Dye Reagent was purchased from Bio-Rad Laboratories (California, USA).