Confocal Imaging of Cellular Peroxide

Confocal microscopy Imaging was performed as described in [24 (link)]. Briefly, cells were cytospun on fibronectin-coated (Cell-tak-coated (Corning, New York, USA)) 35-mm glass-bottom dishes (Ibidi, Gräfelfing, Germany)] and incubated for 20 min at 37 °C with 5 μM DCF-DA (2,7-dichlorofluorescin diacetate) (Molecular Probes, Eugene, Oregon, USA) to detect cellular peroxide. Stained cells were washed with PBS and examined by a Leica (Wetzlar, Germany) TCS SP8 confocal laser scanning microscopy system utilizing appropriate excitation laser beams (images collected using a 60X objective (1.4 NA). Acquisition, storage, and analysis of data were performed with LasX software from Leica or ImageJ 1.48 (Wayne Rasband, NIH, Bethesda, Maryland, USA).