Microglia Depletion from Primary Glial Cultures Enables to Accurately Address the Immune Response of Astrocytes

Abstract

Astrocytes are the most abundant cells in the CNS parenchyma and play an essential role in several brain functions, such as the fine-tuning of synaptic transmission, glutamate uptake and the modulation of immune responses, among others. Much of the knowledge on the biology of astrocytes has come from the study of rodent primary astrocytic cultures. Usually, the culture is a mixed population of astrocytes and a small proportion of microglia. However, it is critical to have a pure culture of astrocytes if one wants to address their inflammatory response. If present, microglia sense the stimulus, rapidly proliferate and react to it, making it unfeasible to assess the individual responsiveness of astrocytes. Microglia have been efficiently eliminated in vivo through PLX-3397, a colony-stimulating factor-1 receptor (CSF-1R) inhibitor. In this work, the effectiveness of PLX-3397 in eradicating microglia from primary mixed glial cultures was evaluated. We tested three concentrations of PLX-3397—0.2 μM, 1 μM and 5 μM—and addressed its impact on the culture yield and viability of astrocytes. PLX-3397 is highly efficient in eliminating microglia without affecting the viability or response of cultured astrocytes. Thus, these highly enriched monolayers of astrocytes allow for the more accurate study of their immune response in disease conditions.

Keywords

• mixed glial cultures
• astrocytes
• microglia
• CSF-1R
• PLX-3397
• C3