Figure 4. Quantification of intracellular ATP concentrations by using BTeam.

(A) Standard curve for calculation of ATP concentration. The BRET ratio values (mean ± SD) of purified BTeam at 37 °C were plotted against ATP concentrations, and the regression model was obtained by using Hill equations (n = 15 measurements/5 independent repetitions). (B) The basal BRET ratios from cells transiently expressing cyt-BTeam. Mean ± SD values of 6 replicate measurements are shown. (C) Comparison of intracellular ATP concentrations determined by BTeam (closed bar) and firefly luciferase (open bar). ATP concentrations based on cyt-BTeam were calculated from data in (B) using the standard curve (A). For ATP determination by firefly luciferase, see Methods section. Mean ± SD values of triplicate measurements are shown. (D,E) Quantification of mitochondrial ATP concentrations. The basal BRET ratio (D) and calculated ATP concentrations (E) of cultured cells transiently expressing mit-BTeam. Mean ± SD values of 6 replicate measurements are shown. (F) Time-lapse measurements of ATP concentrations within the same cell populations. The BRET ratios of HeLa cells stably expressing cyt-BTeam, which were cultured either in control (blue) or starved (red) conditions, were measured at 1 h intervals. The cells were cultured in phenol red-free DMEM containing 5% FBS. At 0 h, starvation was initiated by exchanging the medium with EBSS containing 5% FBS and 1.0 g/L glucose. After 6 h starvation, the medium was re-exchanged to phenol red-free DMEM containing 5% FBS. A representative data set is shown. Error bars represent SD (n = 10 wells in 96 plate). Results from 2 independent experiments worked consistent.