Selective Depletion of Microglia from Cerebellar Granule Cell Cultures Using L-leucine Methyl Ester.
Microglia, the resident immunocompetent cells of the CNS, play multifaceted roles in modulating and controlling neuronal function, as well as mediating innate immunity. Primary rodent cell culture models have greatly advanced our understanding of neuronal-glial interactions, but only recently have methods to specifically eliminate microglia from mixed cultures been utilized. One such technique - described here - is the use of L-leucine methyl ester, a lysomotropic agent that is internalized by macrophages and microglia, wherein it causes lysosomal disruption and subsequent apoptosis(13,14). Experiments using L-leucine methyl ester have the power to identify the contribution of microglia to the surrounding cellular environment under diverse culture conditions. Using a protocol optimized in our laboratory, we describe how to eliminate microglia from P5 rodent cerebellar granule cell culture. This approach allows one to assess the relative impact of microglia on experimental data, as well as 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.
Department of Neurology, University of Washington; jjebelli@uw.edu.
Joseph
J
Jebelli
Therapeutic Innovation Group, School of Life and Medical Sciences, University College London.
Thomas
T
Piers
Department of Neuroinflammation, University College London.
Jennifer
J
Pocock
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D002450:Cell Communication / Q000187:drug effects / Q000502:physiology; D018929:Cell Culture Techniques / Q000379:methods*; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology* / Q000187:drug effects*; D003594:Cytoplasmic Granules / Q000187:drug effects / Q000502:physiology; D007930:Leucine / Q000031:analogs & derivatives* / Q000494:pharmacology; D017628:Microglia / Q000166:cytology* / Q000187:drug effects*; D009474:Neurons / Q000166:cytology / Q000187:drug effects; D051381:Rats
C030365:leucine methyl ester; D007930:Leucine