Lance ultra camp assay kit

The enzyme inhibition assay was performed against human PDE enzymes (PDE1A, PDE3A, PDE4A, PDE4B, PDE4C, PDE4D, and PDE7A; BPS Biosciences, San Diego, CA, United States) according to the manufacturer’s instructions (LANCE Ultra cAMP assay kit; Perkin Elmer, United States). In each well, 5 μl of 3 nM cAMP, 2.5 μl of PDE enzyme (0.1 ng/well), and 2.5 μl of inhibitor solution were added, and incubated at 37°C for 1 h. After incubation, 5 μL each of Eu-cAMP and ULight-anti-cAMP detection reagent supplemented with 1 mM of IBMX were added. The reaction mixture was incubated at 37°C for 1 h. After incubation, emission signals were collected at 665 nm using EnVision Multilable Reader (Perkin Elmer, United States).

HEK-MOR cells (provided by Dr. Ping-Yee Law, University of Minnesota, USA) were cultured in DMEM (GIBCO) supplemented with 10% FBS, 100 units/mL penicillin, 100 μg/mL streptomycin, 400 μg/mL G418, and 2 mM L-glutamine in T-175 tissue culture flasks and harvested with trypsin/EDTA solution (GIBCO). Cells were plated at 72,000 per well under 100 μL/well of DMEM in 96-well, solid-bottom, white plates (GIBCO) and under 50 μL/well of HBSS containing forskolin or 3-isobutyl-1-methylxanthine at final concentrations of 1 μM and 500 μM, respectively. After 30 min of incubation at room temperature, the concentration of cAMP was determined using a LANCE Ultra cAMP Assay kit (Perkin Elmer). Two hours later, plate fluorescence was measured using a Victor 2 plate reader with excitation at 330 nm and emission at 615 nm and 665 nm. The effects of compounds on MOR-mediated inhibition of cAMP production were determined by treating cells with various concentrations of morphine alone or in the presence of the compounds. The results were presented as percent inhibition of forskolin-stimulated cAMP accumulation: [1 − (cAMP_{compounds/forskolin}/cAMP_forskolin)] × 100%.

GBM cell lines were seeded at 38000 cells/well (SB2b) or 35,000 cells/well (PB1, JK2 and WK1) in 96-well matrigel-coated plates and incubated for 24 h at 37°C, 5% CO₂ in humidified incubator in StemPro complete medium. Media was replaced with stimulation buffer (phenol red free F12 media, 0.1% BSA, 0.5 mM IBMX, pH 7.4). Cells were stimulated with agonists (sCT, hCT, rAmy, CGRP or adrenomedullin) at concentrations ranging from 10^− 6 M – 10^− 12 M, or 10^− 5 M forskolin, or vehicle for 30 min at 37 °C. Cells were lysed (0.3% Tween 20, 5 mM Hepes, 0.1% BSA, pH 7.4) and 5 μl of cell lysate from each well was transferred to a corresponding well of 384-well optiplate. Intracellular cAMP levels in the wells were determined using Lance Ultra cAMP assay kit (Perkin Elmer) according to the manufacturer’s instructions and detected using an Envision multilabel 2103 reader. Raw RFU values were converted using a cAMP standard curve to give absolute cAMP concentrations. Data were analysed by three-parameter logistic curve and are presented as percentage of 10^− 5 M forskolin response.