Mouse NSC line C17.2 [33 ] was cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen) containing 2 mM l -glutamine (Invitrogen), 10 % fetal bovine serum (FBS) (Sijiqing Biotech, China), 5 % horse serum (Gibco), 100 units/mL of penicillin, and 100 μg/mL of streptomycin. The cultures were maintained in a standard humidified incubator in 5 % CO2 at 37 °C, with fresh medium replaced every 2 days, and split 1:4 when the cells reached 90 % confluence.
Primary neural stem cells were isolated from newborn SD rat cerebral cortex and cultured in uncoated 25-mL flasks in DMEM/F-12 medium (Invitrogen) containing N2 and B27 supplements (Invitrogen) plus basic fibroblast growth factor (Promega, 20 ng/mL) and epidermal growth factor (Promega, 20 ng/mL). After 5–7 days culture in vitro (DIV), the primary neurospheres were collected and dissociated with 0.05 % trypsin plus 200 mM EDTA for 10 min at 37 °C and mechanically triturated with fire-polished glass pipettes. The single cells were resuspended at a density of 50,000 cells per mL of serum-free medium and cultured for 3–5 days. The number and diameters of neurospheres were assessed as described [34 (link)].
Primary neural stem cells were isolated from newborn SD rat cerebral cortex and cultured in uncoated 25-mL flasks in DMEM/F-12 medium (Invitrogen) containing N2 and B27 supplements (Invitrogen) plus basic fibroblast growth factor (Promega, 20 ng/mL) and epidermal growth factor (Promega, 20 ng/mL). After 5–7 days culture in vitro (DIV), the primary neurospheres were collected and dissociated with 0.05 % trypsin plus 200 mM EDTA for 10 min at 37 °C and mechanically triturated with fire-polished glass pipettes. The single cells were resuspended at a density of 50,000 cells per mL of serum-free medium and cultured for 3–5 days. The number and diameters of neurospheres were assessed as described [34 (link)].
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