BRET-based ATP Biosensor Assay

Cytosolic and mitochondrial ATP levels were quantified as described by Yoshida et al. [31 (link)]. HAP1 WT and TMBIM5 KO cells were transiently transfected with plasmids carrying the bioluminescence energy transfer (BRET)-based ATP biosensor BTeam without targeting the signal sequence (for cyto-ATP determination) or targeted to mitochondria (for mito-ATP determination) using TurboFectin reagent (OriGene). Then, 48 h after transfection, cells were incubated for 30 min in phenol red-free medium supplemented with 30 μM nanoluciferase (NLuc) inhibitor to avoid disturbance from the BTeam released from dead cells. Afterwards, NLuc substrate (Promega) was added to the medium, and the plate was incubated for 20 min. Subsequently, luminescent emissions from the cells were measured at 37 °C at 520/560 nm (Yellow Fluorescent Protein (YFP) emission) and at 430/470 nm (NLuc emission). ATP concentration was calculated as YFP/NLuc emissions ratio. Data are expressed as mitochondrial/cytosolic ATP.