Isolation of murine microglial cells for RNA analysis or flow cytometry

Cardona, Astrid E.; Huang, DeRen; Sasse, Margaret E.; Ransohoff, Richard M.

doi:10.1038/nprot.2006.327

Cited by 220 publications

(193 citation statements)

References 30 publications

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“…SE was induced with pilocarpine in Ccr2 rfp/rfp and control Ccr2 +/+ animals maintained on the FVB background as described earlier. Four days after SE, the mice were perfused with Hanks' buffered salt solution, the brains were removed, and mononuclear cells were separated with a 30%/70% Percoll gradient as previously reported (69,70). Single-cell suspensions from the brain were stained with anti-CD11b-PerCP (M1/70; BioLegend) and anti-Ly6C-FITC (AL-21; BD Pharmingen).…”

Section: Methodsmentioning

confidence: 99%

Infiltrating monocytes promote brain inflammation and exacerbate neuronal damage after status epilepticus

Varvel

Neher

Bosch

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

269

291

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The generalized seizures of status epilepticus (SE) trigger a series of molecular and cellular events that produce cognitive deficits and can culminate in the development of epilepsy. Known early events include opening of the blood-brain barrier (BBB) and astrocytosis accompanied by activation of brain microglia. Whereas circulating monocytes do not infiltrate the healthy CNS, monocytes can enter the brain in response to injury and contribute to the immune response. We examined the cellular components of innate immune inflammation in the days following SE by discriminating microglia vs. brain-infiltrating monocytes. Chemokine receptor 2 (CCR2 + ) monocytes invade the hippocampus between 1 and 3 d after SE. In contrast, only an occasional CD3 + T lymphocyte was encountered 3 d after SE. The initial cellular sources of the chemokine CCL2, a ligand for CCR2, included perivascular macrophages and microglia. The induction of the proinflammatory cytokine IL-1β was greater in FACS-isolated microglia than in brain-invading monocytes. However, Ccr2 knockout mice displayed greatly reduced monocyte recruitment into brain and reduced levels of the proinflammatory cytokine IL-1β in hippocampus after SE, which was explained by higher expression of the cytokine in circulating and brain monocytes in wild-type mice. Importantly, preventing monocyte recruitment accelerated weight regain, reduced BBB degradation, and attenuated neuronal damage. Our findings identify brain-infiltrating monocytes as a myeloid-cell subclass that contributes to neuroinflammation and morbidity after SE. Inhibiting brain invasion of CCR2 + monocytes could represent a viable method for alleviating the deleterious consequences of SE. myeloid cell heterogeneity | epileptogenesis | neuroprotection | seizure | microgliosis S tatus epilepticus (SE) is a serious medical emergency that triggers a series of cellular and molecular events that can result in the development of epilepsy, a chronic neurological disorder characterized by a persistently lowered seizure threshold (1). The early consequences of SE in rodents include a robust neuroinflammatory response, selective neuronal degeneration, and transient opening of the blood-brain barrier (BBB), leading to later cognitive decline. Although the neuroinflammatory features of SE in man are less well known, extravasation of albumin into the brain was observed for patients who died in SE (2), elevated cerebrospinal fluid levels of the cytokines IL-6, IL-8, and CXCL10 are typically found in patients with refractory SE compared with patients with other inflammatory neurologic disorders (3), and intense gliosis (both astrocytes and microglia) was observed in the temporal cortex of a patient with new-onset focal seizures that progressed to refractory SE (4). These admittedly sparse clinical findings are consistent with the much more extensive animal literature in demonstrating a florid inflammatory response of the brain to SE (5). We and others have provided evidence in animal models of epilepsy that quenching inflamma...

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Section: Methodsmentioning

confidence: 99%

Infiltrating monocytes promote brain inflammation and exacerbate neuronal damage after status epilepticus

Varvel

Neher

Bosch

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

269

291

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“…Microglia were isolated as previously described with minor modifications 34 . Rats were decapitated, brains were rapidly removed and placed in Leibovitz-15 media.…”

Section: Methodsmentioning

confidence: 99%

Visualization and genetic modification of resident brain microglia using lentiviral vectors regulated by microRNA-9

et al. 2013

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Functional studies of resident microglia require molecular tools for their genetic manipulation. Here we show that microRNA-9-regulated lentiviral vectors can be used for the targeted genetic modification of resident microglia in the rodent brain. Using transgenic reporter mice, we demonstrate that murine microglia lack microRNA-9 activity, whereas most other cells in the brain express microRNA-9. Injection of microRNA-9-regulated vectors into the adult rat brain induces transgene expression specifically in cells with morphological features typical of ramified microglia. The majority of transgene-expressing cells colabels with the microglia marker Iba1. We use this approach to visualize and isolate activated resident microglia without affecting circulating and infiltrating monocytes or macrophages in an excitotoxic lesion model in rat striatum. The microRNA-9-regulated vectors described here are a straightforward and powerful tool that facilitates functional studies of resident microglia.

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“…The present procedure details an effective method of isolating cortical microglia from neonatal mice resulting in a higher average yield of microglia (7.5 x 10 5 cells/pup cortex) than previously reported methods utilizing digestive enzymes and/or a Percoll gradient (3-5 x 10 5 cells/ whole CNS) 30,33,34 . As evidenced by fluorescent imaging, immunocytochemistry, and ELISA, the microglia isolated via this procedure are capable of undergoing morphological and biochemical responses when exposed to immunogenic stimuli such as Pam and LPS.…”

Section: Discussionmentioning

confidence: 95%

“…However, enzymatic digestion can alter the immunophenotype of the cells by reducing cell-surface antigen expression 32 , and results in lower cell yield per animal than the method described herein. Specifically, we report an average microglia yield per pup cortex of 7.5 x 10 5 cells, while previously reported isolation methods from whole CNS via a Percoll gradient yield 3-5 x 10 5 cells 30,33,34 . The present procedure circumvents the use of digestion enzymes by isolating microglia based on their low-adherence properties, thereby preserving the microglial immunophenotype and functionality.…”

Section: Introductionmentioning

confidence: 90%

“…Therefore, isolation of microglia is a powerful investigative tool into these biological processes, as many in vivo features of microglial activation can be recapitulated in culture. Several methods are available for isolating microglia, including isolation via a Percoll gradient following enzymatic digestion of CNS tissue 30,31 . However, enzymatic digestion can alter the immunophenotype of the cells by reducing cell-surface antigen expression 32 , and results in lower cell yield per animal than the method described herein.…”

Section: Introductionmentioning

confidence: 99%

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Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates

Daniele

Edwards

Maguire‐Zeiss

2014

JoVE

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Isolation of microglia from CNS tissue is a powerful investigative tool used to study microglial biology ex vivo. The present method details a procedure for isolation of microglia from neonatal murine cortices by mechanical agitation with a rotary shaker. This microglia isolation method yields highly pure cortical microglia that exhibit morphological and functional characteristics indicative of quiescent microglia in normal, nonpathological conditions in vivo. This procedure also preserves the microglial immunophenotype and biochemical functionality as demonstrated by the induction of morphological changes, nuclear translocation of the p65 subunit of NF-κB (p65), and secretion of the hallmark proinflammatory cytokine, tumor necrosis factor-α (TNF-α), upon lipopolysaccharide (LPS) and Pam 3 CSK 4 (Pam) challenges. Therefore, the present isolation procedure preserves the immunophenotype of both quiescent and activated microglia, providing an experimental method of investigating microglia biology in ex vivo conditions. Video LinkThe video component of this article can be found at

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Isolation of murine microglial cells for RNA analysis or flow cytometry

Cited by 220 publications

References 30 publications

Infiltrating monocytes promote brain inflammation and exacerbate neuronal damage after status epilepticus

Infiltrating monocytes promote brain inflammation and exacerbate neuronal damage after status epilepticus

Visualization and genetic modification of resident brain microglia using lentiviral vectors regulated by microRNA-9

Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates

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