Abstract
To shorten the time between brain harvesting and microglia isolation, and characterization, we utilized the MACS® neural dissociation kit followed by OctoMACS® CD11b magnetic bead isolation technique to positively select for brain microglia expressing the pan-microglial marker CD11b, a key subunit of the membrane attack complex (MAC). This protocol yields a viable and highly pure (>95%) microglial population of approximately 500,000 cells per pup that is amenable for in vitro characterization within hours or days after being harvested from brain tissue. Primary microglia from C57Bl/6 mice were plated for next-day analyses of morphology and cellular markers by immunocytochemistry or for analysis of gene expression under resting or LPS-stimulated conditions. The ease of isolation enables investigators to perform molecular and cellular analyses without having to wait 1–2 weeks to isolate microglia by conventional methods involving mechanical agitation to dislodge these from astrocyte beds.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Ransohoff RM, Perry VH (2009) Microglial physiology: unique stimuli, specialized responses. Annu Rev Immunol 27:119–145
McGeer PL, McGeer EG (2004) Inflammation and neurodegeneration in Parkinson’s disease. Parkinsonism Relat Disord 10(Suppl 1):S3–S7
Tansey MG, McCoy MK, Frank-Cannon TC (2007) Neuroinflammatory mechanisms in Parkinson’s disease: potential environmental triggers, pathways, and targets for early therapeutic intervention. Exp Neurol 208:1–25
McCoy MK, Martinez TN, Ruhn KA et al (2006) Blocking soluble tumor necrosis factor signaling with dominant-negative tumor necrosis factor inhibitor attenuates loss of dopaminergic neurons in models of Parkinson’s disease. J Neurosci 26:9365–9375
Harms AS, Lee JK, Nguyen TA et al (2012) Regulation of microglia effector functions by tumor necrosis factor signaling. Glia 60(2):189–202
Kurrasch DM, Huang J, Wilkie TM et al (2004) Quantitative real-time polymerase chain reaction measurement of regulators of G-protein signaling mRNA levels in mouse tissues. Methods Enzymol 389:3–15
Acknowledgement
The content of this article was adapted from one that appeared in the [Date] issue of the Miltenyi Biotec newsletter. This work was supported by NIH/NINDS grant 5R011NS049433 and The Michael J. Fox Foundation for Parkinson’s Research.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer Science+Business Media New York
About this protocol
Cite this protocol
Harms, A.S., Tansey, M.G. (2013). Isolation of Murine Postnatal Brain Microglia for Phenotypic Characterization Using Magnetic Cell Separation Technology. In: Joseph, B., Venero, J. (eds) Microglia. Methods in Molecular Biology, vol 1041. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-520-0_5
Download citation
DOI: https://doi.org/10.1007/978-1-62703-520-0_5
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-519-4
Online ISBN: 978-1-62703-520-0
eBook Packages: Springer Protocols