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

Stefano G. Daniele; Amanda A. Edwards; Kathleen A. Maguire-Zeiss

doi:10.3791/51005

Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neon...

JoVE Journal
Immunology and Infection

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JoVE Journal Immunology and Infection

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

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09:12 min

January 30, 2014

DOI: 10.3791/51005-v

Stefano G. Daniele¹, Amanda A. Edwards¹, Kathleen A. Maguire-Zeiss¹

¹Department of Neuroscience,Georgetown University Medical Center

One key to successful investigation of microglial biology is the preservation of microglial immunofunction ex vivo during isolation from CNS tissue. Isolating microglia via rotary shaking results in highly pure and immunofunctional cell cultures as assessed by fluorescent imaging, immunocytochemistry, and ELISA following microglia activation with the proinflammatory stimuli lipopolysaccharide (LPS) and Pam₃CSK₄ (Pam).

The overall goal of this procedure is to isolate cortical microglia from murine neonates. This is accomplished by first micros dissecting cortical brain tissue from Muren neonates. The second step is to grow mixed glial cell cultures for 17 to 21 days in vitro.

Next, the microglia are isolated from these mixed glial cultures. The final step is to plate microglial cells in cell culture plates for further experimentation. Ultimately, immunofluorescence microscopy and Eliza are used to show that this isolation protocol preserves microglial immuno phenotype and functionality.

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