Light induced cytotoxicity can be prevented with new culture medium. (a) Viability of 7 d.i.v. β-III+ cortical neurons ± light at indicated dose in NEUMO (in replacement of Neurobasal) and SOS (in replacement of B27) using PI exclusion assay. (b) Number of PI positive cells in 7 d.i.v. hippocampal neuronal cultures ± optogenetic stimulation at 1 Hz in control media conditions (Neurobasal + B27) with additional antioxidants (AA) and in NEUMO + SOS. Note: the protection of cortical and hippocampal neurons during treatment with light only in NEUMO + SOS media conditions. (c) Representative images and quantification of GFAP+ astrocyte numbers in 7 d.i.v. cortical neuron enriched cultures after ± light at indicated dose in NEUMO + SOS. (d) Non-linear fitted plots from Sholl Analysis of astrocytes kept in the dark (black line: 62 cells analyzed) or exposed to light (blue line: 56 cells analyzed) and p value calculated from two tailed unpaired t-test of mean intersection number. All above histograms are normalized to controls (with the exception of b) and data represents means ± s.e.m. of a number of biological replicates (value within or above 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. Light doses in kJ/m2 are shown above histograms and as insets within representative images of irradiated experiments. Cell culture media conditions are described above images and under respective histograms.