Incucyte zoom live imaging system

Live-cell imaging using the IncuCyte ZOOM live imaging system (Essen BioScience) was started immediately after plating iNs after differentiation at DIV 5 in 96 well plate, assigning 4 fields per well, 6 wells per genotype for each of three independent differentiations. Neurite length and neurite branch point were measured using the Essen BioScience neurite analysis tool after imaging every 4 h for 3 days.

The SH-SY5Y cells were plated in 96-well microplates (Greiner, Sigma Aldrich, Sydney, Australia) at 5000 cells per well and then cultured for 7 days. To induce differentiation of the cells, the culture media was replaced with differentiation media (DMEM/F12, 1% PS, 1% FBS, with or without 10 μM all-trans retinoic acid (RA, R2625, Sigma Aldrich)) 24 h after plating, followed by 100% media changes every 48 h. Dimethyl sulfoxide (DMSO) was used as a vehicle control for the RA, and the vehicle control medium was also changed every 48 h. SH-SY5Y cells were treated with different intensities of AC or DC MF (either with or without RA) for 2 days, starting 24 h after plating, and the cells were cultured until day 7 without any further exposure to EMF from day 3. Cell images (four images per well of a 96-well plate) were collected every day using an IncuCyte Zoom live imaging system (Essen Bioscience, Michigan, USA) and analyzed by the IncuCyte software to acquire data on cell confluence (area occupied by cells over the total area), neurite length (total neurite length in mm mm⁻²), and number of neurite branch points (number per mm²). Controls were carried out using cells cultured in 10% FBS media. Mean data were calculated from technical replicates of 5 wells for the DC MF and 4 wells for the AC MF. Mean data ± SEM are shown from three independent experimental repeats.

Cardiac fibroblasts were plated at 80% confluency and incubated overnight (37 °C, 5% CO₂). Vehicle (DMSO), Yoda1 (10 μm), or staurosporine (1 μm) were applied the following day, and cells were incubated for 24 h prior to commencing the live/dead cell viability assay (Thermo Fisher Scientific) according to the manufacturer's instructions. Cells were imaged using the IncuCyte ZOOM Live Imaging System (Essen Bioscience). The total number of fluorescent cells in each well was calculated using built-in algorithms, using an average from 9 images/well of a 12-well plate. The mean data are shown as the number of live cells relative to the total number of cells.