75 nm nylon mesh

Primary mixed glial cell cultures were established as described previously (42 (link)). Briefly, the brain cells of 1- to 3-day-old neonatal C57BL/6 mice were dissociated by repeated pipetting and then passed through a 75-nm nylon mesh (Corning, NY, USA). The cells were subsequently washed once in cold PBS and cultured in DMEM (with high glucose) supplemented with 10% FBS and 1% penicillin–streptomycin. The medium was changed on days 3, 5, and 7 for the astrocytes and on day 3 only for the microglia. On day 10, the flasks were shaken at 260 rpm for 2 h to remove any non-adherent cells (mainly microglia). The remaining adherent astrocytes were detached with trypsin-EDTA and then plated again for further experiments. The purity of the astrocyte cultures was greater than 95%.

Duck neurons were isolated according to our previously described method for isolating mouse neurons (29 (link)). Briefly, the brain was collected from 9-day-old duck embryos, the meninges were peeled away and carefully removed, and the cortex was transferred into a new dish filled with HBSS, and then cut into 1-mm pieces by using scissors; the shears were transferred into 0.1% trypsin diluted in HBSS and digested for 20 min at RT. The trypsin was removed by transferring the brain cells into a new tube with proper DMEM, and then DNase I was added and treated for 5 min at RT. The DNase I was removed and the cells were collected by centrifugation at 1,200 rpm for 10 min at RT. Then, the collected cells were resuspended with DMEM and dissociated by repeated pipetting (<15 times). The cells were passed through a 75-nm nylon mesh (Corning, NY, USA) to separate the single cells, washed once in HBSS, and then cultured in D-polylysine–pretreated plates in DMEM supplemented with 5% FBS and 1% penicillin–streptomycin for 6 h. Finally, the cells were washed once with phosphate-buffered saline (PBS), and the medium was replaced with serum-free, neural-basal medium supplemented with 2% B-27 PLUS (Gibco-BRL, NY, USA), and the cells were incubated for 5 days to form a monolayer.

Primary mixed glial cell cultures were established as described previously [26 (link)]. Briefly, brain tissues from 2-day-old BALB/c mice were dissociated by repeated pipetting and then passed through a 75-nm nylon mesh (Corning, NY, USA). The cells were washed once in cold PBS and cultured in DMEM (with high glucose) supplemented with 10% FBS and 1% penicillin–streptomycin. The medium was changed on days 3, 5, and 7. On day 10, the flasks were shaken at 260 rpm for 2 h to remove any non-adherent cells (mainly microglia). The remaining adherent astrocytes were detached with trypsin-EDTA and then plated again for further experiments. The purity of the isolated astrocytes for further studies was greater than 95%, which was examined by immunohistochemistry using the anti-GFAP antibody.