Primary microglia from neonatal Sprague-Dawley rats (Crl:CD) were isolated by the method of Giulian and Baker [
15] as previously described [
16]. Briefly, brains were freed from meninges and dissociated mechanically and enzymatically with 0.1 % trypsin (Biochrom, Merck Millipore, Darmstadt, Germany) and 0.25 % DNase (Roche Diagnostic GmbH, Mannheim, Germany). Cells were plated at 1–2 brains per poly-
l-lysine (Sigma-Aldrich, Munich, Germany) coated culture flasks (25 cm
2; 75 cm
2; Sarstedt, Nümbrecht, Germany) and cultivated in Dulbecco’s Modified Eagle Medium (DMEM; life technologies, Carlsbad, USA) supplemented with 10 % fetal calf serum (FCS; Biochrom, Merck Millipore, Darmstadt, Germany) and 1 % penicillin/streptomycin (life technologies, Carlsbad, USA; culture medium referred to as: MGP
+), where indicated culture flasks were treated with granulocyte macrophage-colony stimulating factor (GM-CSF; 5 ng/ml; Peprotech; Hamburg, Germany). After 7–8 days, loosely attached microglia were harvested from mixed glial cell cultures by shaking for 30–45 min on an orbital shaker-incubator (Edmund Bühler, Hechingen, Germany) at 37 °C. Cells in the supernatant were re-plated at 3–6 × 10
4 cells per 12-mm glass coverslip in 24-well plates for immunocytochemistry staining, directly resuspended in 500 μl MGP
+ for BrdU flow cytometry, 5 × 10
5 per well in 6-well plates for reverse transcription polymerase chain reaction (RT-PCR), or 2.5 × 10
5 in 12-well plates (all Nunc, Life Technologies, Paisley, UK) for phagocytosis and flow cytometry analysis. The remaining astrocytes were washed with PBS and harvested with 0.25 % trypsin/0.05 % EDTA. After isolation, the astrocytes were incubated with an APC-conjugated mouse/rat anti-GLAST (ACSA-1) antibody and with FITC-conjugated anti-BrdU antibody.