Co-culturing Immune Cells and Mouse-Derived Mixed Cortical Cultures with Oxygen-Glucose Deprivation to In Vitro Simulate Neuroinflammatory Interactions After Stroke

Studying interactions between neural cells and glial cells in vitro remains an essential tool for scientists worldwide, and with the addition of oxygen–glucose deprivation (OGD) can be particularly useful for identifying mechanisms related to ischemic stroke-related injury and repair. In developing these protocols in the lab, however, we discovered the limitation of co-culturing immune cells with pure neuronal cultures as the standard media for immune cells impair neuronal growth and vice versa. Thus, we optimized a mixed cortical cell culture system that does not require the use of glial-conditioned media to support the viability and growth of neurons but can nonetheless be used to quantify neuronal survival and dendritic arborization. The following methods provide a guide as to how to culture mixed cortical cells from mouse pups (postnatal day 0–2). Additionally, we demonstrate how to co-culture mixed cortical cells with immune cells (e.g., B cells) to study neuro-immune interactions in vitro.