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

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