Ham s f 12 nutrient mix medium

Human S1P₁-expressing CHO cells (catalog number: 93-0207C2) were purchased from Eurofins DiscoverX (Fremont, CA, USA)[15 (link)], and were cultured in Ham’s F-12 nutrient mix medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific), 100 U/mL penicillin, 100 μg/mL streptomycin, 300 μg/mL hygromycin B and 800 μg/mL geneticin. The cAMP Gi Kit (Cisbio) was used for the cAMP assay. The test substance was dissolved in 80% ethanol containing 10 mM NaOH and diluted in Hanks' balanced salt solution (HBSS; Life Technologies) containing 0.1% fatty acid-free bovine serum albumin (BSA; Sigma-Aldrich). The cells were seeded in 96-well half-area plates (1.65 × 10⁴ cells in 25 μL, per well) with 1 mM 3-isobutyl-1-methylxanthine. A 20-μL volume of test substance solution and 5 μL of 10 μM forskolin solution were added, and the cells were incubated for 45 minutes at room temperature. Subsequently, homogenous time-resolved fluorescence reagents (Cisbio) were added to the plate and incubated for 1 hour at room temperature. The ratio of the acceptor and donor emission signals (ratio = signal 665 nm/signal 615 nm x 10⁴) was measured in each individual well using an EnVision plate reader (PerkinElmer). The average was taken from three independent experiments with each experiment run in duplicate and expressed as a percentage of the maximum response to S1P.

Polyphenolic compounds, 3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT), 2, 2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) disodium salt (ABTS), fluorometric intracellular ROS kit, penicillin/streptomycin, and 2-(4-amidinophenyl)-6-indolecarbami dine dihydrochloride (DAPI) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Parishin derivatives were bought from Chengdu Biopurify Phytochemicals Ltd. (Chengdu, Sichuan, China). Fetal bovine serum (FBS) was obtained from Young in Frontier Company (Seoul, Korea). 2,2-Diphenyl-1-picrylhydrazyl (DPPH) was bought from MedChemExpress (Princeton, NJ, USA). Ham’s F-12 Nutrient Mix medium, BCA protein assay kit, and cDNA synthesis kit (ReverTra Ace qPCR RT Kit) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Eagle’s Minimum Essential Medium (EMEM) was obtained from ATCC (Manassas, VA, USA). H₂O₂ was purchased from Fujifilm Wako Pure Chemical (Osaka, Japan). The SYBR green qPCR Kit was supplied by TOYOBO (Osaka, Japan). Lactate dehydrogenase (LDH) cytotoxicity assay kit was bought from TAKARA (Shiga Prefecture, Japan). Clarity western chemiluminescent (ECL) substrate kit was supplied by Bio-Rad Laboratories (Hercules, CA, USA).