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Here, we demonstrate that it is possible to achieve high\u2010yield isolation of pure microglial (MAC\u20101<jats:sup>+<\/jats:sup>\/Fcrls<jats:sup>+<\/jats:sup>\/Ccr2<jats:sup>\u2010<\/jats:sup>) 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\u2010supplemented 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\u2010dependent. 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\u201310 ng\/ml) treatment by secreting pro\u2010inflammatory cytokines such as TNF\u2010\u03b1, IL\u20106, IL\u20101\u03b2 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\u2010CSF, 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. 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