Effects of Mava and OM on fluorescence lifetime in IANBD-cTnC ^T53C exchanged myofibrils in a mock screen illustrates Z′ quality of high-throughput assays. (A) IANBD-cTnC lifetime is reduced with Mava in the presence of ATP at low (left) and high (right) Ca²⁺. OM causes small but significant increases in lifetime at low and high Ca²⁺, ***P < 0.0001, *P < 0.002. Data is mean ± SD; n = 23 repeats in wells from one experimental preparation. (B) In the presence of ATP at low Ca²⁺, Mava reduces lifetime as compared to DMSO control and 3× SD of the Mava data is well separated from 3× SD of the DMSO data (blue vs. grey: Z′ = 0.19). For OM, 3× SD of the OM data overlap with 3× SD of the DMSO data (green vs. grey: Z′ = −2.71). In the mock screen (n = 23 wells for each of two conditions) the average for each condition is indicated with a dashed line and 3× SD above and below the average is shown with a solid line. Note that dashed green (mean + OM) line is coincident with the upper solid grey line (+3× SD +DMSO).