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Isolation of Microglia from Mouse or Human Tissue.

ABSTRACT: Microglia are the innate immune cells of the central nervous system. Although numerous methods have been developed to isolate microglia from the brain, the method of dissociation and isolation can have a profound effect on the function of these highly dynamic cells. Here, we present an optimized protocol to isolate CD11b+ cells (microglia) from mouse or human brain tissue using magnetic bead columns. Isolated microglia can be used to model diseases with neuroinflammatory components for potential therapeutic discoveries. For complete details on the use and execution of this protocol, please refer to Hanamsagar et al. (2017), Rivera et al. (2019), and Edlow et al. (2019).

SUBMITTER: Bordt EA

PROVIDER: S-EPMC7416840 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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Isolation of Microglia from Mouse or Human Tissue.

Bordt Evan A EA Block Carina L CL Petrozziello Tiziana T Sadri-Vakili Ghazaleh G Smith Caroline J CJ Edlow Andrea G AG Bilbo Staci D SD

STAR protocols 20200603 1

Microglia are the innate immune cells of the central nervous system. Although numerous methods have been developed to isolate microglia from the brain, the method of dissociation and isolation can have a profound effect on the function of these highly dynamic cells. Here, we present an optimized protocol to isolate CD11b+ cells (microglia) from mouse or human brain tissue using magnetic bead columns. Isolated microglia can be used to model diseases with neuroinflammatory components for potential ...[more]

PMID: 32783030

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Project description:BACKGROUND:Microglia play key roles in neuron-glia interaction, neuroinflammation, neural repair, and neurotoxicity. Currently, various microglial in vitro models including primary microglia derived from distinct isolation methods and immortalized microglial cell lines are extensively used. However, the diversity of these existing models raises difficulty in parallel comparison across studies since microglia are sensitive to environmental changes, and thus, different models are likely to show widely varied responses to the same stimuli. To better understand the involvement of microglia in pathophysiological situations, it is critical to establish a reliable microglial model system. METHODS:With postnatal mouse brains, we isolated microglia using three general methods including shaking, mild trypsinization, and CD11b magnetic-associated cell sorting (MACS) and applied RNA sequencing to compare transcriptomes of the isolated cells. Additionally, we generated a genome-wide dataset by RNA sequencing of immortalized BV2 microglial cell line to compare with primary microglia. Furthermore, based on the outcomes of transcriptional analysis, we compared cellular functions between primary microglia and BV2 cells including immune responses to LPS by quantitative RT-PCR and Luminex Multiplex Assay, TGFβ signaling probed by Western blot, and direct migration by chemotaxis assay. RESULTS:We found that although the yield and purity of microglia were comparable among the three isolation methods, mild trypsinization drove microglia in a relatively active state, evidenced by high amount of amoeboid microglia, enhanced expression of microglial activation genes, and suppression of microglial quiescent genes. In contrast, CD11b MACS was the most reliable and consistent method, and microglia isolated by this method maintained a relatively resting state. Transcriptional and functional analyses revealed that as compared to primary microglia, BV2 cells remain most of the immune functions such as responses to LPS but showed limited TGFβ signaling and chemotaxis upon chemoattractant C5a. CONCLUSIONS:Collectively, we determined the optimal isolation methods for quiescent microglia and characterized the limitations of BV2 cells as an alternative of primary microglia. Considering transcriptional and functional differences, caution should be taken when extrapolating data from various microglial models. In addition, our RNA sequencing database serves as a valuable resource to provide novel insights for appropriate application of microglia as in vitro models.

Dataset Information

Isolation of Microglia from Mouse or Human Tissue.

Publications

Isolation of Microglia from Mouse or Human Tissue.

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