Epmotion 96 liquid handling workstation

The screening studies were performed in 96-well assay plates (Corning 3590 flat bottom, transparent) and each library was screened at a final concentration of 25μM. An epMotion^® 96 liquid handling workstation (Eppendorf) was used to dispense homogenate and NAADP into assay plates. Fluo-3 fluorescence was monitored using a Tecan Infinite M1000 Pro plate reader. For all screening experiments, fluo-3 fluorescence changes were monitored in the presence of compound for 35 cycles (6 minutes) prior to the addition of an EC₉₀ concentration of NAADP (167nM final concentration). For the LOPAC^®1280 library, 0.25ul of vehicle (DMSO) or compound (10mM) was dispensed into the assay plates using a LabCyte ECH0550 acoustic nanoliter dispensing system. The assay was started by addition of 99.75μl of sea urchin egg homogenate. For experiments screening the Selleck GPCR compound library, baseline fluo-3 fluorescence of the homogenate (97.5ul) was monitored for 1.5 minutes prior to the addition of 2.5ul vehicle (DMSO) or compound (1mM) using the epMotion^® 96. Z’ values were calculated to assess separation of distributions of positive and negative controls, as described elsewhere [45 (link)].