Fig. 3.

Quantification of CD39- and CD73-dependent ATP degradation and adenosine generation by T_reg and OvCA cells. a 10⁴ cells/well were cultured in 100 μl complete RPMI 1640 medium with or without addition of 1 μM ATP and/or 100 μM of either ARL67156 or APCP or solvent control. After 24 h, free extracellular ATP was measured via addition of recombinant firefly luciferase and d-Luciferin (in excess). A standard curve ranging from 0.1 nM to 10 μM ATP confirmed that the bioluminescent signal obtained under these conditions was directly proportional to the level of available ATP (r = 0.99). b Primary OvCA cells were cultured with and without addition of ARL67156 before ATP levels from supernatant were determined as in (a). As primary cells were limited in number, the APCP control had to be omitted. c, d Adenosine generation from T_reg, OvCA cell lines (c) and ascites-derived OvCA cells (d) was quantified using a reporter gene assay based on RIP1-CRE-luc⁺ pRL-CMV⁺ ADORA2A⁺ HEK-293 reporter cells that are co-incubated with the cells of interest at a 1:1 ratio (10⁴ cells/well from each cell type). Adenosine in the cellular microenvironment binds to the ectopically expressed ADORA2A on HEK-293 cells, which leads to increased cAMP levels in the ‘sensor’ cells and thus enhanced activity of the cAMP responsive RIP1-Cre-luc reporter. Firefly luciferase activity was measured after 4 h co-incubation, normalised for co-transfected pRL-CMV activity and related to extracellular adenosine concentrations via a co-determined standard curve. Where indicated, the specific inhibitors of CD39 and CD73, ARL67156 and APCP were added (100 μM) (n = 3). Please note the different scales