Confluent flasks of CHO-OPRD1 cells were collected with TrypLE Express, and resuspended in F-12 media supplemented with 10% FBS and 25 mM HEPES, at a density of 6.67e5 cells per ml and plated (3 μl per well) into white solid TC-treated 1,536-well plates (Corning, NY). Plates were incubated overnight at 37 °C in a 5% CO2-humidified incubator. The next day, 1 μl of increasing concentrations of naloxone (4 × final concentration in assay buffer) were added to separate columns of the assay plates containing cells. Next, increasing concentrations of BMS-986187 (40 nl of 100 × final concentration in 100% DMSO) were added to separate rows of the assay plates by acoustic dispense using an Echo-550 (Labcyte, Sunnyvale, CA) from Echo-qualified 1,536-well source plates (Labcyte). Plates were covered with a lid and incubated at room temperature for 90 min. Incubations were terminated by the addition of 2 μl PathHunter Reagent. One hour later luminescence was detected using a Viewlux imaging plate reader (PerkinElmer). All PathHunter detection reagents were purchased from DiscoveRx, other chemicals, unless otherwise stated, were from Sigma.