ATP Efflux Assay for Transfected HEK293 Cells

The ability of the transfected HEK293 cells to release ATP into the culture medium was determined using confluent monolayers as described previously [14 (link),19 (link)]. In brief, the medium was removed and replaced with 50 μL efflux buffer, consisting of 11.5 mM HEPES (pH 7.4), 130 mM NaCl, 5 mM MgCl2, 1.5 mM CaCl2, and 11.5 mM glucose. The cells were then incubated for 1 hr at 27 °C. Next, 50 µL BactiterGlo reagent (Promega) dissolved in efflux buffer was added to each well. Bioluminescence was subsequently determined in real time in a Flex Station 3 microplate reader (Molecular Devices, San Jose, CA, USA) as detailed previously [14 (link),19 (link)]. The real-time ATP efflux assay was run at 27 °C for the first 1 h and then at 37 °C for 2 hr. The initial low temperature allowed endogenous ecto-nucleotidases to degrade the Abcc6-independent background ATP efflux induced by the medium change.