High‐yield isolation of murine microglia by mild trypsinization

Abstract

Microglia can be isolated with high purity but low yield by shaking off loosely adherent cells from mixed glial cultures. Here we describe a new technique for isolating microglia with an average yield close to 2,000,000 microglial cells/mouse pup, more than five times higher than that of the shaking method. Confluent mixed glial cultures are subjected to mild trypsinization (0.05-0.12%) in the presence of 0.2-0.5 mM EDTA and 0.5-0.8 mM Ca2+. This results in the detachment of an intact layer of cells containing virtually all the astrocytes, leaving undisturbed a population of firmly attached cells identified as >98% microglia. These almost pure microglial preparations can be kept in culture for weeks and show proliferation and phagocytosis. Treatment with macrophage colony-stimulating factor and lipopolysaccharide, alone or in the presence of interferon gamma, induces typical microglial responses in terms of proliferation, morphological changes, nuclear factor-kappaB translocation, NO, and tumor necrosis alpha release and phagocytosis. This method allows for the preparation of highly enriched mouse or rat microglial cultures with ease and reproducibility. Because of its high yield, it can be especially convenient when high amounts of microglial protein/mRNA are required or in cases in which the starting material is limited, such as microglial cultures from transgenic animals.

IGF‐1 receptor‐mediated ERK/MAPK signaling couples status epilepticus to progenitor cell proliferation in the subgranular layer of the dentate gyrus

“…At 3-days post-plating, 85% of cells were microglia, as assessed by double labeling with Hoecht and CD11b. This value approximates the microglial purity reported by Saura et al (2003).…”

“…Mixed microglia cultures were prepared using the method described by Saura et al (2003) with minor modification. Briefly, mixed glial cultures were prepared from cerebral cortices of 1-day-old C57Bl/6 mice.…”

PI3Kδ inhibition reduces TNF secretion and neuroinflammation in a mouse cerebral stroke model

“…To harvest microglia, 490% confluent glia cultures were rinsed with FBS-free DMEM:F12 and incubated with 0.05% trypsin for 20 min. Microglia remained adherent, whereas other glial types lifted away 52 . To harvest astrocytes, 470% confluent glia cultures were incubated with 0.25% trypsin for 2 min.…”

“…The dissociated tissue was then passed through culture medium, which was either Neurobasal medium (Life Technologies) supplemented with B-27 (Life Technologies) and 1% L-glutamine (v/v) for neuronal enrichment, or DMEM (Life Technologies) containing 50% F12 (v/v; Life Technologies), 10% heat-inactivated FBS (Life Technologies) and 1% L-glutamine for glial enrichment. Cells were dissociated by trituration and seeded in either Neurobasal/B-27/L-glutamine or DMEM:F12/FCS/L-glutamine medium at densities from 10 5 cells ml À 1 (for neurons) to 1.5 Â 10 6 cells ml À 1 (for glia) 52 . All cells were maintained at 37°C in humidified 5% CO 2 /95% air.…”

Probucol inhibits LPS-induced microglia activation and ameliorates brain ischemic injury in normal and hyperlipidemic mice

“…Microglial cells were isolated from brains of 2 or 3-d-old C57BL/6J mice as previously described with some modification [27] . In brief, mixed glial cultures were prepared from the cerebral cortex, and mechanical and chemical dissociations (0.5% trypsin-EDTA: Gibco, Waltham, MA, USA) were performed.…”