Primary microglia isolation from mixed cell cultures of neonatal mouse brain tissue.

Microglia are the main resident immunological cells of the central nervous system (CNS) that are functionally equivalent to macrophages. However, due to the cellular heterogeneity of the brain, it is technically challenging to obtain highly specific, healthy microglia with the desired phenotype in sufficient yield for in vivo experiments. This study presents a new and easy method for the isolation of microglia cells from mouse pups (P1-P3). This method consists of a 20-day protocol, divided in three sections: mixed cell culture, culture maintaining (astrocytes growing), and isolation after astrocytes confluence. This procedure produces microglia with no astrocyte, neuron and oligodendrocyte precursors cells contamination that are functionally active to answer inflammatory responses based on the measurement of cell and inflammatory markers. This technique requires approximately three hours for the isolation of neonatal mixed cell culture, 20 to 22 days for microglia growing and two days before starting experiments from pure and healthy microglia. This study presents an isolation protocol that is adapted from existing methods and is economic, rapid, not tedious, with little manipulation time and work. This method also allows to isolate large amount of high specific microglia cells with no specific phenotype and with great reproducibility and efficiently. This study provides a detailed description of the methods that is routinely used in our laboratory for the isolation and the culture of microglia, with emphasis on the steps that are deemed most critical for obtaining large amount of pure and healthy cultures.