title: A simple magnetic separation method for high-yield isolation of pure primary microglia.
abstract: Microglial cells play a dynamic role in the brain beyond their established function of immune surveillance. Activated microglia play key roles in neural development, neuroinflammation, neural repair and neurotoxicity. They are particularly important in several neurodegenerative diseases in which sustained microglial activation contributes to the progression of neurodegenerative processes. Consequently, understanding microglial function in CNS health and disease has become an area of active research in recent years. However, a significant obstacle to progress in this field has been the inherent difficulties in obtaining large amounts of primary microglial cells to routinely perform mechanistic studies and characterize signaling pathways regulating the dynamics of microglial activation. Herein, we describe a novel column-free magnetic separation protocol for high-yield isolation of primary microglia from mouse postnatal mixed glial cultures. The procedure is based on optimized culture conditions that enable high microglial cell densities in confluent mixed glial cultures followed by highly efficient recovery of pure microglia by magnetic separation. The novel column-free magnetic separation system utilizes tetrameric antibody complexes (TAC) with dual specificity for CD11b-PE labeled microglia and dextran magnetic nanoparticles. An FcR blocker (anti-CD16/32) is added to enhance the purity of the microglial separation by preventing non-specific labeling of other cell types. This procedure yields on average >3×10⁶ microglial cells per mouse pup, with a remarkable purity of 97% and recovery of around 87% of microglia from the mixed glial population. Importantly, the microglia obtained by this method are fully functional and respond like cells obtained by conventional isolation techniques.
authors.affiliation: Department of Biomedical Sciences, Iowa Center for Advanced Neurotoxicology, Iowa State University, Ames, IA 50011, USA.
authors.forename: Richard
authors.initials: R
authors.lastname: Gordon
authors.forename: Colleen E
authors.initials: CE
authors.lastname: Hogan
authors.forename: Matthew L
authors.initials: ML
authors.lastname: Neal
authors.forename: Vellareddy
authors.initials: V
authors.lastname: Anantharam
authors.forename: Anumantha G
authors.initials: AG
authors.lastname: Kanthasamy
authors.forename: Arthi
authors.initials: A
authors.lastname: Kanthasamy
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D000831:Animals, Newborn; D001921:Brain / Q000166:cytology; D039481:CD11b Antigen / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002469:Cell Separation / Q000295:instrumentation / Q000379:methods*; D002478:Cells, Cultured; D016207:Cytokines / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods; D005786:Gene Expression Regulation / Q000187:drug effects; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D008280:Magnetics / Q000295:instrumentation / Q000379:methods*; D051379:Mice; D008810:Mice, Inbred C57BL; D008840:Microfilament Proteins; D017628:Microglia / Q000378:metabolism / Q000502:physiology*; D009569:Nitric Oxide / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D051844:alpha-Synuclein / Q000494:pharmacology
chemical_list: C491049:Aif1 protein, mouse; D039481:CD11b Antigen; D002135:Calcium-Binding Proteins; D016207:Cytokines; D005904:Glial Fibrillary Acidic Protein; D008070:Lipopolysaccharides; D008840:Microfilament Proteins; D017382:Reactive Oxygen Species; D051844:alpha-Synuclein; D009569:Nitric Oxide