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Isolation of murine microglial cells for RNA analysis or flow cytometry

Nature Protocols volume 1, pages 1947–1951 (2006)Cite this article

Abstract

There is increasing interest in the isolation of adult microglia to study their functions at a morphological and molecular level during normal and neuroinflammatory conditions. Microglia have important roles in brain homeostasis, and in disease states they exert neuroprotective or neurodegenerative functions. To assay expression profiles or functions of microglia, we have developed a method to isolate microglial cells and infiltrating leukocytes from adult mouse brain. This protocol uses a digestion cocktail containing collagenase and dispase, and it involves separation over discontinuous percoll gradients. Isolated cells can be used for RNA analysis, including RNase protection analysis (RPA), quantitative RT-PCR, high-density microarray, proteomic or flow cytometric characterization of cell surface markers or adoptive transfer. Cell isolation can be completed in less than 4 h.

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Figure 1: Procedure workflow.
Figure 2: Flow cytometry analysis of cells recovered from 70%–37%interphase.
Figure 3: RNA integrity and RPA results.

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Acknowledgements

This work was supported by the National Institutes of Health (NS32151 to R.M.R.), the Dana Foundation (to R.M.R.) and the National Multiple Sclerosis Society (FG 1528-A-1 and TA 3021-A-1 to A.E.C.).

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Authors and Affiliations

  1. Department of Neurosciences, Neuroinflammation Research Center, Lerner Research Institute, Cleveland Clinic, Cleveland, 44195, Ohio, USA

    Astrid E Cardona, DeRen Huang, Margaret E Sasse & Richard M Ransohoff

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  1. Astrid E Cardona
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  2. DeRen Huang
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  3. Margaret E Sasse
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  4. Richard M Ransohoff
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Correspondence to Richard M Ransohoff.

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Cardona, A., Huang, D., Sasse, M. et al. Isolation of murine microglial cells for RNA analysis or flow cytometry. Nat Protoc 1, 1947–1951 (2006). https://doi.org/10.1038/nprot.2006.327

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