Embryonic Brain Cell Isolation and Analysis

Dissected embryonic E14.5 brains were titrated in FACS buffer (PBS - 1% Fetal calf serum - 0.02% NaN₃) using pipette tip, and filtered through 70-µm cell strainers (Fisher Scientific, Hampton, NH). Single cell suspensions were blocked with 10% normal rat serum on ice for 30 min., and stained with a combination of anti-mouse cell surface flow cytometric markers CD11b-FITC (BioLegend, San Diego, CA), GLAST-PE, and O4-APC (Miltenyi Biotec, Bergish Gladbach, Germany) at 4 °C in dark for 1 h with continuous rotation. The stained cells were then washed, centrifuged, and resuspended into 1 mL of FACS buffer, and acquired on a two-laser, six-color Gallios cytometer (Beckman Coulter, Pasadena, CA). The threshold for FACS analysis was set based on the surface expression of each individual marker in the samples as compared to isotype control using IgG-stained cells in the same channel, and Flow cytometric data were analyzed with the Kaluza 1.3 software (Beckman Coulter, Brea, CA), as reported [35 (link),36 (link)]. Cell populations were calculated as the percentages among the total cells.