Cellstar cell culture plate

Cortices were isolated from at E10.5 embryos in glucose enriched L-15 and mechanically dissociated in cold PBS. The cells were spun down and suspended in DMEM-F12 supplemented with GlutaMAX, non-essential amino acids, penicillin–streptomycin, pyruvate, B27 minus Vitamin A, N2 (Life Technologies), 10 ng ml⁻¹ basic fibroblast growth factor and 20 ng ml⁻¹ epidermal growth factor (R&D System) in low cell-binding 96-well round-bottom plates (Sigma). Neurospheres were dissociated to single cells every 4 days using TrypLE (Gibco). Cells were counted and plated in 12-well Cellstar cell culture plates (Greiner) at a density of 150,000 cells per well. To ascertain the neural identity of neurospheres, cells were plated on 12 mm poly-D-lysine-coated round coverslips (Corning) in 24-well Cellstar cell culture plates (Greiner) at a density of 50,000 cells per well. After 4 days, cells were fixed for 15 min in 2% paraformaldehyde in PBS, rinsed in PBS, blocked in 5% normal goat serum, 0.3% Triton X-100/PBS for 20 min and incubated overnight at 4 °C in blocking buffer containing mouse anti-Nestin (1:1,000; Millipore). Cells counterstained with DAPI. Coverslips were mounted with Mowiol.

The 10⁶ CD14⁺ monocytes isolated by positive selection were resuspended into 2 mL of RPMI medium supplied with 10% heat-inactivated foetal calf serum (FCS), 1% L-glutamine, and 1% penicillin/streptomycin. They were incubated in 6- or 12-well CELLSTAR^® cell culture plate (Cat #657970 and #665970, respectively, Greiner Bio-One GmbH, Frickenhausen, Germany) at 37 °C with 5% CO₂ supplement. This special plate with a cell-repellent surface prevents cell attachment. After 24 h of incubation, the cells were harvested by centrifugation at 300× g for 10 min and resuspended into new working RPMI medium at the concentration of 1 × 10⁶ cell/mL.