Clariostar fluorescence plate reader

The PACE-YFP, HSE-YFP, and PDRE-YFP reporter genes were integrated into the LEU2 locus of the yeast genome. Cells were induced by the addition of 960 ng/ml ATc for 16 h in galactose-containing media. As positive controls the empty vector sample shifted to 37 °C for 16 h (PACE and HSE) and 4 h induction of b₂-DHFR (PDRE) were used. 4 OD₆₀₀ of cells were harvested by centrifugation (12,000 × g, 5 min, RT) and resuspended in 400 µl H₂O. About 100 µl of the cell suspension were transferred to flat-bottomed black 96-well imaging plates (BD Falcon, Heidelberg, Germany) in technical triplicates. Cells were sedimented by gentle spinning (30 g, 5 min, RT), and fluorescence (excitation 497 nm, emission 540 nm) was measured using a ClarioStar Fluorescence plate reader (BMG-Labtech, Offenburg, Germany). The corresponding wild-type strain not expressing YFP was used for background subtraction of autofluorescence. Fluorescence intensities were normalized to the value obtained from the wild-type empty vector control in each of three independent biological replicates.

H₂O₂ concentrations in PAM were quantified using a fluorometric Hydrogen Peroxidase Assay kit (Sigma–Aldrich Co., Ltd). This kit uses horseradish peroxidase and a red fluorescent peroxidase substrate (λex: 540 nm, λem: 590 nm) and allows quantification of H₂O₂ in a range of concentrations between 0 and 10 µM. PAM were diluted 1/50 before each measurement in order to achieve an adequate concentration of H₂O₂. Calibration curves (see Fig. 14) were plotted for the different liquid media used for the preparation of PAM from an initial 3% hydrogen peroxide solution to avoid any influence of medium absorption on the fluorescence measurements. Samples in black 96-well plates were analyzed using a CLARIOstar fluorescence plate reader (BMG LABTECH) at room temperature.Figure 14

Calibration curves of hydrogen peroxide concentration in three solvents.

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.