Screenworks peak pro software

At day 7 or day 14 of iPSC-CPCs differentiation, or at day 10 of iPSC-CMs maintenance, intracellular Ca²⁺ oscillation was studied on a FLIPR Tetra system (Molecular Devices, Berkshire, UK) using FLIPR Calcium 5 Assay Kit (Molecular Devices) as described by Pointon et al.^{55 (link)}. Briefly, the cell culture media were first removed and replaced with 25 µl/well of fresh culture medium 2 h prior to assay. The cells were loaded with 1 × FLIPR Calcium 5 dye at 37 °C for 40 min by adding 25 µl/well of 2 × FLIPR Calcium 5 dye. The cell plates were transferred to a FLIPR Tetra system and maintained at 37 °C. Ca²⁺ oscillations were recorded at Ex 485 nm/Em 530 nm with 0.05 s exposure time/read and 0.12 s read interval. 450 reads were collected at the basal condition, followed by a further 350 reads post-the addition of 12.5 µl/well of 10 µM Isoproterenol solution (prepared from 10 mM stock, I5627, Sigma-Aldrich, diluted in assay medium) to reach a final concentration of 2 µM isoproterenol using onboard liquid handling within the FLIPR Tetra, allowing both pre- and post- compound reads to be obtained from the same well from plates maintained at 37 °C during the recording period. Ca²⁺ oscillation peak frequencies and peak amplitude were analyzed using the ScreenWorks Peak Pro Software (Molecular Devices).

FLIPR Tetra^® data were analyzed using ScreenWorks PeakPro Software (Molecular Devices). Spatial uniformity correction was enabled, and peak detection was utilized with the following parameters: smooth width = 5, fit width = 10, and dynamic amplitude threshold = 20. Each well was normalized to its baseline reading before compound addition. Data were graphed using GraphPad Prism version 7. In the primary screen, compounds were counted as hits only if both replicates were greater or less than 2 times the standard deviation of the DMSO treated wells. Significant differences between vehicle and compound treated wells were determined by one-way ANOVA followed by Dunnett’s test. GraphPad Prism version 7 was used to perform all statistical analyses.