title: Validation of Flow Cytometry and Magnetic Bead-Based Methods to Enrich CNS Single Cell Suspensions for Quiescent Microglia.
abstract: Microglia are resident mononuclear phagocytes within the CNS parenchyma that intimately interact with neurons and astrocytes to remodel synapses and extracellular matrix. We briefly review studies elucidating the molecular pathways that underlie microglial surveillance, activation, chemotaxis, and phagocytosis; we additionally place these studies in a clinical context. We describe and validate an inexpensive and simple approach to obtain enriched single cell suspensions of quiescent parenchymal and perivascular microglia from the mouse cerebellum and hypothalamus. Following preparation of regional CNS single cell suspensions, we remove myelin debris, and then perform two serial enrichment steps for cells expressing surface CD11b. Myelin depletion and CD11b enrichment are both accomplished using antigen-specific magnetic beads in an automated cell separation system. Flow cytometry of the resultant suspensions shows a significant enrichment for CD11b(+)/CD45(+) cells (perivascular microglia) and CD11b(+)/CD45(-) cells (parenchymal microglia) compared to starting suspensions. Of note, cells from these enriched suspensions minimally express Aif1 (aka Iba1), suggesting that the enrichment process does not evoke significant microglial activation. However, these cells readily respond to a functional challenge (LPS) with significant changes in the expression of molecules specifically associated with microglia. We conclude that methods employing a combination of magnetic-bead based sorting and flow cytometry produce suspensions highly enriched for microglia that are appropriate for a variety of molecular and cellular assays.
authors.affiliation: Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE, 68198, USA.
authors.forename: T A
authors.initials: TA
authors.lastname: Volden
authors.affiliation: Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE, 68198, USA.
authors.forename: C D
authors.initials: CD
authors.lastname: Reyelts
authors.affiliation: Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE, 68198, USA.
authors.forename: T A
authors.initials: TA
authors.lastname: Hoke
authors.affiliation: Developmental Neuroscience, Munroe-Meyer Institute for Genetics and Rehabilitation, University of Nebraska Medical Center, Omaha, NE, 68198, USA.
authors.forename: J
authors.initials: J
authors.lastname: Arikkath
authors.affiliation: Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE, 68198, USA. sbonasera@unmc.edu.|University of Nebraska Medical Center, 3028 Durham Research Center II, Omaha, NE, 68198-5039, USA. sbonasera@unmc.edu.
authors.forename: S J
authors.initials: SJ
authors.lastname: Bonasera
mesh_terms: D000818:Animals; D002469:Cell Separation; D002531:Cerebellum / Q000166:cytology*; D005434:Flow Cytometry / Q000379:methods*; D007031:Hypothalamus / Q000166:cytology*; D018189:Immunomagnetic Separation / Q000379:methods*; D051379:Mice; D008810:Mice, Inbred C57BL; D017628:Microglia / Q000166:cytology*; D060888:Real-Time Polymerase Chain Reaction; D013535:Suspensions
chemical_list: D013535:Suspensions
keywords: Magnetic bead-based cell separation; Microglia, flow cytometry; Microglia, purification; RT-qPCR