Developer toolbox 1

Cell metabolic activity was measured by resazurin reduction. Briefly, 10% (v/v) of resazurin (0.3 mg/ml) was added to the cells and incubated for 20 h. The fluorescence of resorufin, resulting from the reduction of resazurin by metabolically active cells, was measured at λ_ex = 530 nm and λ_em = 590 nm in Synergy^TM Mx (BioTek, Winooski, VT, USA).
For the evaluation of membrane permeability, cells were incubated with a mixture of a cell-permeable nuclear dye Hoechst 33342 (1:12000, Invitrogen, Eugene, OR, USA), and a membrane-impermeable dye SYTOX^TM Green (1:45000, Thermo Fisher Scientific, Waltham, MA, USA), for 20 minutes at 37 °C and 7% CO₂. Cells were visualized in a 37 °C, CO₂ atmosphere with a 20× Nikon objective in a high-throughput automated fluorescence wide-field microscope (IN Cell Analyzer 2000, GE Healthcare, Little Chalfont, UK). The acquired images were then analyzed with Developer Toolbox 1.9.2 (GE Healthcare).

ROS production was evaluated using 2′,7′-dichlorofluorescein diacetate (DCFDA), as described previously^{40 (link)}. The oxidation of the dye by ROS generates a highly fluorescent compound, 2,7-dichlorofluorescein (DCF) that can be detected at λ_ex = 504 nm and λ_em = 529 nm. Briefly, cells were incubated with DCFDA (5 µM) for 30 minutes, washed with warm PBS/5% FBS, and then imaged in a 37 °C, CO₂ atmosphere with a 20× Nikon objective in a high-throughput automated fluorescence wide-field microscope (IN Cell Analyzer 2000, GE Healthcare, Little Chalfont, UK). The acquired images were then analyzed with Developer Toolbox 1.9.2 (GE Healthcare).
Lipid peroxidation was measured with BODIPY®, following the manufacturer’s instructions (Thermo Fisher Scientific). Upon oxidation by lipid hydroperoxides, BODIPY® changes its maximum fluorescence emission wavelength from 590 nm to 510 nm. Briefly, cells were incubated with BODIPY® (10 µM) for 30 minutes, washed with PBS, and then visualized in a 37 °C, CO₂ atmosphere with a 20× Nikon objective in a high-throughput automated fluorescence wide-field microscope (IN Cell Analyzer 2000, GE Healthcare). The acquired images were analyzed with CellProfiler^TM.