High Content In Vitro Survival Assay of Cortical Neurons

Neuronal survival in vitro is usually used as a parameter to assess the effect of drug treatments or genetic manipulation in a disease condition. Easy and inexpensive protocols based on neuronal metabolism, such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), provide a global view of protective or toxic effects but do not allow for the monitoring of cell survival at the single neuronal level over time. By utilizing live imaging microscopy with a high-throughput microscope, we monitored transduced primary cortical neurons from 7-21 days in vitro (DIV) at the single neuronal level. We established a semi-automated analysis pipeline that incorporates data stratification to minimize the misleading impact of neuronal trophic effects due to plating variability; here, we provide all the necessary commands to reproduce it. Key features • The protocol enables monitoring of primary cortical neuron survival from DIV 7 to 21 in 96-well plates following various cellular treatments. • It provides single-cell and real-time imaging resolution, enabling the identification of small changes in viability over time. • It provides a detailed description of semi-automated neuronal detection over time. • It relies on data stratification based on the neuronal starting number, which helps reduce the impact of neuronal trophic effects due to plating variability. • It has been used to assess the effect of glial extracellular vesicles on cortical neurons, as reported in [1].