4 2 hydroxyethyl 1 piperazineethanesulfonic acid hepes buffer

BM cells were isolated from C57BL/6 J female mice by conventional methods^{35 (link)} and suspended at 1.0 × 10⁶ cells/mL in DC culture medium composed of complete RPMI 1640, 10% heat-inactivated FCS (Invitrogen, CA, USA), 2.5 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (Invitrogen), 1 mM sodium pyruvate (Invitrogen), 50 μM 2-mercaptoethanol (Invitrogen), 1% non-essential amino acids (NEAA, Invitrogen), 100 U/mL of penicillin, and 100 μg/mL of streptomycin (Sigma, MO, USA). The cells were cultured in the presence of 100 ng/mL of Flt-3L (R&D systems, MN, USA) and test extracts, fractions, and compounds were maintained at 37 °C in a 5% CO₂ atmosphere. In some assays, BM cells were treated with sample on day 6. On day 7, the cells were stimulated with 5 ng/mL of lipopolysaccharide (LPS) (Sigma). After overnight stimulation, the cells were treated with antibodies, and the expression of cell surface markers was evaluated by flow cytometry using a FACSCanto II instrument BD Biosciences, CA, USA). The production of cytokines IL-12p40 and IL-10 in the supernatant of cell cultures after LPS stimulation was measured by using ELISA kits (BD Pharmingen). In the phase of screening step, each sample was evaluated repeatedly in the two wells, and after identification of 14-DHE, samples at each concentration were evaluated in the three wells.

The transfected Chinese hamster ovary (CHO) cells, rat basophil leukemia (RBL) cells, U373 cells and BA/F3 cells were obtained from Eurofins Scientific (Le Bois I’Eveque, France). Various buffers namely Hank’s balanced salt solution (HBSS) buffer, Dulbecco’s modified Eagle medium (DMEM) buffer and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer were obtained from Invitrogen (Carlsbad, CA, USA). The reference agonists and/or antagonists atropine sulphate salt, acetylcholine chloride, [Sar9, Met(O2)11]-Substance P, [Arg8]-vasopressin (AVP), (+)butacamol, clozapine, dopamine, [d(CH2)5 1, Tyr(Me)2]-AVP, serotonin, SCH 2330, L-733,060, (S)-WAY-100635 and 3-isobutyl-1-methylxanthine (IBMX) were purchased from Sigma-Aldrich (St. Louis, MO, USA). All the other chemicals and reagents used were purchased from E. Merck, Fluka (Rupert-Mayer-Str., Munich, Germany), and Sigma-Aldrich (St. Louis, MO, USA), unless otherwise stated, and were of highest grade available.

Recombinant mouse granulocyte-macrophage colony stimulating factor (rmGM-CSF) and recombinant mouse interleukin (rmIL)-4 were purchased from JW Creagene (Korea). Roswell Park Memorial Institute (RPMI) 1640 medium, fetal bovine serum (FBS), and penicillin/streptomycin were obtained from Gibco (Grand Island, USA). Lipopolysaccharide (LPS) from Escherichia coli O111:B4 (ultra-pure grade) was obtained from InvivoGen (USA). V450-conjugated anti-CD11c, fluorescein isothiocyanate (FITC)-conjugated anti-CD80, anti-IL-10, anti-CD4, annexin V/propidium iodine (PI) kit, phycoerythrin (PE)-conjugated anti-CD86, anti-IL-12p70, allophycocyanin (APC)-conjugated tumor necrosis factor (TNF)-α, PE-Cy7-conjugated anti-CD11c, anti-CD8, BV510-conjugated live/dead kit, and enzyme linked immunosorbent assay (ELISA) kits for TNF-α, IL-12p70, IL-10, IFN-γ, IL-2, and IL-5 were purchased from BD biosciences (USA). APC-conjugated anti-MHC-I, PE-Cy7-conjugated anti-MHC-II, anti-CD8, 2-mercaptoethanol, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, Minimum Essential Medium (MEM) non-essential amino acids, and CellTrace Violet Cell Proliferation Kit were purchased from Invitrogen (USA).

Mulberrofuran G (1), kuwanon G (2), and albanol B (3) were isolated and identified from the root bark of M. alba Linn following a method described previously [63 (link)]. The purity of these compounds was considered to be >98% as evidenced by spectral data. A MAO-GloTM assay kit was purchased from Promega (Promega Corporation, Madison, WI, USA). Transfected Chinese hamster ovary (CHO) cells were obtained from Eurofins Scientific (Le Bois I’Eveque, France). Hank’s balanced salt solution (HBSS), Dulbecco’s modified Eagle medium, and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer were obtained from Invitrogen (Carlsbad, CA, USA). The hMAO isozymes and reference drugs selegiline, DA, serotonin, butaclamol, SCH 23390, clozapine, and (S)-WAY-100635 were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Human endogenous (U373MG cells), murine interleukin-3 dependent pro-B (Ba/F3) and a transfected Chinese hamster ovary (CHO) cell lines were obtained from Eurofins Scientific (Le Bois I’Eveque, France). Buffers—Dulbecco’s modified Eagle medium (DMEM) buffer, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer and Hank’s balanced salt solution (HBSS) buffer—were purchased from Invitrogen (Carlsbad, CA, USA). The reference agonists dopamine, [Sar9, Met(O2)11]-SP, serotonin and arginine vasopressin, and antagonists (+) butaclamol, L 733060, (S)-WAY-100635, and [d(CH₂)5¹,Tyr(Me)₂]-AVP) were obtained from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals and reagents were purchased from Merck and Fluka, unless otherwise stated and were of highest available grade.