Light induces cytotoxicity in vitro. (a) Blue light (470 ± 10 nm) LED setup with control unit regulating delivery of power, pulse duration and frequency of light to cells in 6 well dish. (b) Viabilities of cortical neurons (β–III-tubulin+ (β–III)) of 7 or 21 days in vitro (d.i.v.) using propidium iodide (PI) exclusion assay after ± light at indicated light dose (units of kJ/m2). (c) Mixed glia (astrocytes (GFAP+) and OPCs (NG2+)) viabilities determined using PI exclusion assay after ± light treatment. (d) Representative images and quantification of GFAP+ astrocyte numbers in 7 d.i.v. cortical neuron enriched cultures after ± light at indicated dose. (e) Example of astrocyte morphological tracings after ± light treatment using GFAP staining and NeuronJ tracing and concentric radii of 10 µm steps overlaid in green to aid the reader. Sholl intersection masks with heat map of intersection number inset, generated using Sholl Analysis software from NeuronJ tracings. (f) Non-linear fitted plots of data from Sholl Analysis of astrocytes kept in the dark (black line: 58 cells analyzed) or exposed to light (blue line: 76 cells analyzed) and p value calculated from two tailed unpaired t-test of mean intersection number. (g) Microglial numbers (IB4+ cells) and volumes from binarised image masks of IB4+ cells using ImageJ and expressed as percentage area after ± light. (h) Representative images and quantification of NG2+ cell viabilities using PI exclusion assay in OPC enriched cultures after ± light treatment at 108 kJ/m2 light dose. All above histograms are normalized to controls with data representing means ± s.e.m. of a number of biological replicates (n indicated on each histogram) and p values from Student’s two tailed unequal variance t-test. Black and blue histograms represent conditions kept in the dark or exposed to light respectively, and dashed lines are control values. Media conditions for experiments are described above their respective images. Light doses in kJ/m2 are shown above histograms and as insets within representative images of cells treated with light.