| Calcium mobilization assays | Human mGluR4/Gqi5/CHO cells (30,000 cells/20 •l/well) are plated in blackwalled, clear-bottomed, TC treated, 384 well plates in DMEM containing 10% dialyzed FBS, 20 mM HEPES, 100 units/ml penicillin/streptomycin, and 1 mM sodium pyruvate. The cells are grown overnight at 37 °C in the presence of 5% CO2. During the day of assay, the medium is replaced with 20 μL of 1 μM Fluo-4, AM prepared as a 2.3 mM stock in DMSO and mixed in a 1:1 ratio with 10% (w/v) pluronic acid F-127 and diluted in Assay Buffer (Hank’s balanced salt solution, 20 mM HEPES and 2.5 mM Probenecid) for 45 minutes at 37 °C. Dye is removed and replaced with 20 μL of Assay Buffer. Test compounds are transferred to daughter plates using an Echo acoustic plate reformatter and then diluted into Assay Buffer. Ca2+ flux is measured using the Functional Drug Screening System 6000. Baseline readings are taken (10 images at 1 Hz, excitation, 470±20 nm, emission, 540±30 nm) and then 20 •l/well test compounds are added using the FDSS’s integrated pipettor. Cells are incubated with compounds for approximately 2.5 minutes and then an EC20 concentration of glutamate is applied; 2 minutes later an EC80 concentration of glutamate is added. For concentration-response curve experiments, compounds are serially diluted 1:3 into 10 point concentration response curves and are transferred to daughter plates using the Echo. Test compounds are again applied and followed by EC20 concentrations of glutamate. For fold shift experiments, compounds are added at 2X their final concentration and then increasing concentrations of glutamate are added in the presence of vehicle or the appropriate concentration of test compound. Curves are fitted using a four point logistical equation using Microsoft XLfit. Subsequent confirmations of concentrationresponse parameters are performed using independent serial dilutions of source compounds and data from multiple days experiments are integrated and fit using a four point logistical equation in GraphPad Prism. Calcium assays are used to assess activity of compounds at mGluRs 1, 4 and 5. |
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