title: Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain.
abstract: Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed. Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.
authors.affiliation: Ischemia Research Unit, Fraunhofer Institute for Cell Therapy and Immunology.
authors.forename: Claudia
authors.initials: C
authors.lastname: Pösel
authors.affiliation: Ischemia Research Unit, Fraunhofer Institute for Cell Therapy and Immunology.
authors.forename: Karoline
authors.initials: K
authors.lastname: Möller
authors.affiliation: Ischemia Research Unit, Fraunhofer Institute for Cell Therapy and Immunology; Fraunhofer Research Institution for Marine Biotechnology, University of Lubeck.
authors.forename: Johannes
authors.initials: J
authors.lastname: Boltze
authors.affiliation: Ischemia Research Unit, Fraunhofer Institute for Cell Therapy and Immunology.
authors.forename: Daniel-Christoph
authors.initials: DC
authors.lastname: Wagner
authors.affiliation: Ischemia Research Unit, Fraunhofer Institute for Cell Therapy and Immunology; Department of Neurology, University of Leipzig; gesa.weise@izi.fraunhofer.de.
authors.forename: Gesa
authors.initials: G
authors.lastname: Weise
mesh_terms: D000818:Animals; D001921:Brain / Q000276:immunology / Q000473:pathology; D002452:Cell Count; D002469:Cell Separation / Q000379:methods; D002499:Centrifugation, Density Gradient; D004195:Disease Models, Animal; D004210:Dissection / Q000379:methods; D004660:Encephalitis / Q000276:immunology / Q000473:pathology; D005434:Flow Cytometry / Q000379:methods; D020244:Infarction, Middle Cerebral Artery / Q000276:immunology / Q000473:pathology*; D007962:Leukocytes / Q000276:immunology*; D008297:Male; D008810:Mice, Inbred C57BL; D017628:Microglia / Q000276:immunology / Q000473:pathology; D009186:Myelin Sheath / Q000276:immunology; D020521:Stroke / Q000276:immunology / Q000473:pathology; D013536:Suture Techniques