Related Experiment Video
Updated: Mar 17, 2026
Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates
Published on: January 30, 2014
A simplified approach for efficient isolation of functional microglial cells: Application for modeling
Julia E Sepulveda-Diaz1, Mohand O Ouidja2, Sergio B Socias1,3
1Institut National De La Santé Et De La Recherche Médicale, U 1127, CNRS, Unité Mixte De Recherche (UMR) 7225, Sorbonne Universités, UPMC Univ Paris 06, UMR S 1127, Institut Du Cerveau Et De La Moelle Epinière, ICM, Paris, France.
Abstract:
Purified microglial cells in culture are frequently used to model brain inflammatory responses but obtaining large yields of these cells on a routine basis can be quite challenging. Here, we demonstrate that it is possible to achieve high-yield isolation of pure microglial (MAC-1(+) /Fcrls(+) /Ccr2(-) ) cells from postnatal brain tissue through a simple culture procedure that mainly relies on the adhesion preference of these cells to the polycation polyethyleneimine (PEI) in serum-supplemented DMEM medium. Accordingly, other synthetic or biological substrates failed to mimic PEI effects under the same culture conditions. Replacement of DMEM by DMEM/F12 nutrient mixture did not permit microglial cell isolation on PEI coating, indicating that PEI effects were context-dependent. Remarkably, the lack of culture feeding during progression of microglial cell isolation strongly improved cell yield, suggesting that nutritional deprivation was required to optimize this process. When generated in large culture flasks coated with PEI, cultures of microglial cells were easily recovered by trypsin proteolysis to produce subcultures for functional studies. These cultures responded to lipopolysaccharide (LPS, 1-10 ng/ml) treatment by secreting pro-inflammatory cytokines such as TNF-α, IL-6, IL-1β and by generating nitric oxide and reactive oxygen species. Most interestingly, this response was curtailed by appropriate reference drugs. Microglial cells were also strongly responsive to the mitogenic cytokine GM-CSF, which confirms that the functional repertoire of these cells was well preserved. Because of its high yield and simplicity, we believe that the present method will prove to be especially convenient for mechanistic studies or screening assays. GLIA 2016;64:1912-1924.
Insights
Researchers developed a high-yield method for isolating pure microglial cells using polyethyleneimine (PEI) coating and nutritional deprivation. This technique simplifies obtaining these crucial cells for brain inflammatory response studies.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- Purified microglial cells are vital for studying brain inflammation.
- Current methods for isolating microglial cells yield insufficient quantities for routine research.
- A need exists for a simple, high-yield method for microglial cell isolation.
Purpose of the Study:
- To develop a high-yield, simple method for isolating pure microglial cells from postnatal brain tissue.
- To characterize the functional responses of isolated microglial cells.
- To assess the utility of the isolated microglial cells for mechanistic studies and screening assays.
Main Methods:
- Utilized polyethyleneimine (PEI) coated culture flasks in DMEM medium.
- Employed a nutritional deprivation strategy during cell isolation.
- Verified microglial purity using MAC-1, Fcrls, and Ccr2 markers.
Main Results:
- Achieved high-yield isolation of pure microglial cells (MAC-1(+) /Fcrls(+) /Ccr2(-)).
- PEI coating and nutritional deprivation were critical for isolation success.
- Isolated microglial cells exhibited robust inflammatory responses to LPS and responded to GM-CSF.
Conclusions:
- The developed PEI-based method offers a simple and high-yield approach for microglial cell isolation.
- Isolated microglial cells retain essential functions for inflammatory studies.
- This method is highly suitable for mechanistic studies and drug screening assays.
