A versatile tool for the analysis of neuronal survival

To understand the principles that govern mechanisms of neuronal survival or death it is necessary to systematically model these processes. Methods involving overexpression or knockdown of a gene of interest using non-viral transfection of primary neurons can easily be adapted to study cell death pathways in primary neurons. However, common biochemical approaches to measure cell death are insufficient to measure neuronal viability in these systems. To investigate the functional role of genes in cultured neurons, we therefore established a cell-based assay using a cotransfection/cocultivation approach in primary cortical neurons from mouse or rat. Using this method, it is possible to use well-established cell culture models of neuronal damage, and to analyze cell survival in genetically different neurons on a single-cell basis following apoptotic stimuli under identical conditions. The duration of the entire protocol is 10 days. Finally, the method may be applicable to a wide range of damage models, primary cells, and cell lines as well as it can be used for high content screening (HCS) studies and downstream image cytometry.