Oxidative stress and mitochondrial membrane potential (MitoMP) were measured for the human stem cell-derived neurons with a reactive oxygen species (ROS) detection assay (Abcam) and a MitoMP assay (Cell Signaling). Cells were cultured as described above at a density of 7000 cells per well in white polystyrene flat-bottomed 96-well plates [58 (link)]. The cells were stimulated with 0.1, 1.0, 10.0 or 100.0 ng/ml TNFα and untreated cells served as control. For the ROS assay, after 24 hrs of stimulation the cells were incubated for 60 min at 37°C with the ROS Red Dye working solution. Thereafter, some cells were treated with 10 μM or 20 μM DMSO for 15 min as positive controls whereas the fluorescence signal (Ex490/Em620, bottom optics) was measured against medium (wells without cells) using a BMG fluostar Omega Plate reader (BMG Labtech).
For the MitoMP assay, after 24 hrs of stimulation the cells were incubated with 2 μM JC-1 for 15 min. Some non-stimulated cells were incubated with 50 μM CCCP for 5 min before adding JC-1 serving as positive controls. Thereafter cells were washed with PBS and added PBS before measuring the fluorescence signal (Ex490/Em520) against medium (wells without cells) using a BMG fluostar Omega Plate reader (BMG Labtech).
For the MitoMP assay, after 24 hrs of stimulation the cells were incubated with 2 μM JC-1 for 15 min. Some non-stimulated cells were incubated with 50 μM CCCP for 5 min before adding JC-1 serving as positive controls. Thereafter cells were washed with PBS and added PBS before measuring the fluorescence signal (Ex490/Em520) against medium (wells without cells) using a BMG fluostar Omega Plate reader (BMG Labtech).