Selective depletion of microglia from cerebellar granule cell cultures using L-leucine methyl ester

Abstract

Microglia, the resident immunocompetent cells of the CNS, play multifaceted roles in modulating and controlling neuronal function, as well asmediating innate immunity. Primary rodent cell culture models have greatly advanced our understanding of neuronal-glial interactions, but onlyrecently have methods to specifically eliminate microglia from mixed cultures been utilized. One such technique – described here – is the useof L-leucine methyl ester, a lysomotropic agent that is internalized by macrophages and microglia, wherein it causes lysosomal disruption andsubsequent apoptosis13,14. Experiments using L-leucine methyl ester have the power to identify the contribution of microglia to the surroundingcellular environment under diverse culture conditions. Using a protocol optimized in our laboratory, we describe how to eliminate microglia fromP5 rodent cerebellar granule cell culture. This approach allows one to assess the relative impact of microglia on experimental data, as wellas determine whether microglia are playing a neuroprotective or neurotoxic role in culture models of neurological conditions, such as stroke,Alzheimer’s or Parkinson’s disease.