Characterization of an improved procedure for the removal of microglia from confluent monolayers of primary astrocytes

Cultures of astrocytes can be readily established and are widely used to study the biological functions of these glial cells in isolation. Unfortunately, contamination by microglia can confound results from such studies. Herein, a simple and highly effective modification of a common procedure to remove microglia from astrocyte cultures is described. After becoming confluent, astrocytes were exposed to a mitotic inhibitor for 5-6 days then treated with 50-75 mM L-leucine methyl ester (LME) for 60-90 min. Unlike previous protocols that employed lower LME concentrations on subconfluent cultures or during passage of astrocytes, this protocol effectively depleted microglia from high-density astrocyte monolayers. This was evidenced by the selective depletion of microglial-specific markers. Purified monolayers appeared morphologically normal 24 h after LME treatment and expressed nitric oxide synthase-2 (NOS-2) and cyclooxygenase-2 (COX-2) proteins upon stimulation with LPS plus IFNγ, albeit to a lower level than unpurified monolayers. This difference could be attributed to removal of contaminating microglia from monolayers and not to astrocyte dysfunction, since LME treatment did not alter global protein synthesis and a reactive phenotype could be induced in the purified monolayers. Thus, this modified protocol selectively depletes microglia from high-density primary astrocyte monolayers without compromising their functional integrity.