The effect of drugs on roGFP2-Orp1 oxidation was quantified as described [10 (link)] in the NSCLC cell line H838 stably expressing roGFP2-Orp1 (with or without the mitochondrial targeting sequence). The day before the measurement cells were seeded into a black clear-bottomed 96-well imaging plate (Falcon, Cat No. 353219) at a density of 20,000 cells/well in 200 μL Fluorobrite medium (2% FBS, 25 mM HEPES, 100 U/mL penicillin, and 100 μg/mL streptomycin, 2% Glutamax). A non-transduced control was included on the same plate for background subtraction. In order to obtain the fluorescence intensity values for a fully oxidized and reduced probe, control wells were treated with 2 mM diamide or 10 mM DTT for 15 min at 37 °C. After the entire plate was measured for 8 cycles in a CLARIOstar fluorescence plate reader, BMG Labtech (which allows the simultaneous detection of the two excitation maxima of roGFP2 (400 nm and 485 nm) when emission is monitored at 520 nm), 22 μL of 10x concentrated drug was added and measurement continued for up to 340 min. The readout of the roGFP2 measurement was expressed as the degree of sensor oxidation (OxD, see equation in Ref. [11 (link)]). All treatments were performed in technical triplicate seeded in different quadrants of the imaging plate, to avoid position effects.
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