| Assay Method Information | |
| | Intracellular Calcium Assay |
| Description: | Cell Lines and Cell Culture.The PathHunter U2OS HTR2C β-Arrestin cell line (5-HT2cR-U2OS; DiscoveRx) stably express the nonedited human 5-HT2CR isoform (h5-HT2CR). The 5-HT2CR-U2OS cells were grown in Assay Complete U2OS Medium 31 (DiscoveRx) at 37° C., 5% CO2 and 85% relative humidity according to manufacturer's recommendations utilizing AssayComplete Cell Detachment Reagent (DiscoveRx). Cells were passaged at 70-80% confluence and all experiments were conducted using cells in log phase growth.Intracellular Calcium Assay.The ability of the molecules to act as agonists to induce 5-HT2CR-mediated intracellular calcium (Cai ++) release was conducted in an U2OS cell line stably expressing the human 5-HT2CR. For all molecules examined, the observed potency was shifted rightward relative to 5-HT or WAY163909 (Table 1).Intracellular calcium (Cai ++) release was monitored using the FLIPR Calcium 4 Assay Kit (Molecular Devices) according to previously published protocols with minor modifications. See, e.g., Shashack, M. J., et al., ACS Chem Neurosci. 2 (11), 640-644 (2011); Seitz, P. K.; Bremer, N. M., et al., BMC Neurosci. 13, 25 (2012).Cells were plated at 5,000-7,000 cells/well in Assay Complete Cell Plating Reagent 16 (DiscoveRx) in black-sided, clear bottomed 96-well tissue culture plates and allowed to adhere overnight. Medium was removed and replaced with 40 μl Hank's balanced salt solution without calcium, magnesium and phenol red (HBSS; Corning) plus 40 μl Calcium 4 dye solution in Buffer B supplemented with 2.5 mM probenecid (Sigma-Aldrich) to inhibit extracellular dye transport. Plates were incubated for 60 min at 37° C. followed by 30 min at room temperature in the dark.Fluorescence (λex=485 nm, λem=525 nm) was measured using a FlexStation3 (Molecular Devices). Baseline was established for 17 secs before addition of 20 μl vehicle (HBSS without calcium or magnesium) or 5× concentrated compound. Addition of 5-HT, WAY163909, or ligand occurred at the 17-sec time point and fluorescence was recorded every 1.7 sec for 120 sec. Maximum peak height was determined using FlexStation software (SoftMax Pro 5.4). After the final readings, cells were fixed in 2% paraformaldehyde overnight.Data AnalysisPeak responses from each well were normalized to total cell mass as determined with crystal violet staining. See e.g., Ding, C. et al., ACS Chem. Neurosci 3 (7), 538-545 (2012). |
| Affinity data for this assay | |
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