Knockout dmem f 12

Monocyte Medium: RPMI 1640 (Gibco), 10% FCS (Gibco), 10 mM Hepes (Gibco), 2 mM Glutamax (Gibco), 1% Antibiotic/Antimycotic (Gibco); StemPro Differentiation Medium (SP): KnockOut DMEM/F-12 (Gibco), 2% StemPro Neural Supplement (Gibco), 2 mM Glutamax (Gibco), 1% Antibiotic/Antimycotic (Gibco); OPC Differentiation Medium (OPC Diff): OPC Spontaneous Differentiation Media Kit (Sigma-Aldrich SCM106); OPC Expansion Medium: Human OPC Expansion Culture Media Kit (Sigma-Aldrich SCM107);
Neural Differentiation Medium (NDM): Neurobasal Medium (Gibco), 2% B27 Supplement (Gibco), 2 mM Glutamax (Gibco), 1% Antibiotic/Antimycotic (Gibco); B27 Medium: KnockOut DMEM/F-12 (Gibco), 2% B27 Supplement (Gibco), 2 mM Glutamax (Gibco), 1% Antibiotic/Antimycotic (Gibco); N2 Medium: KnockOut DMEM/F-12 (Gibco), 1% N2 Supplement (Gibco), 2 mM Glutamax (Gibco), 1% Antibiotic/Antimycotic (Gibco); KO DMEM/F-12 Medium: KnockOut DMEM/F-12 (Gibco), 2 mM Glutamax (Gibco); 1% Antibiotic/Antimycotic (Gibco); RPMI 1640 Medium: RPMI 1640 (Gibco), 2 nM Glutamax (Gibco), 1% Antibiotic/Antimycotic (Gibco).

NSCs derived from the human pluripotent stem cell line H9 were purchased from Thermo Fischer Scientific. NSCs were stained with the NSC markers NESTIN, SOX1 and SOX2.
NSCs were washed with cold PBS (without Ca²⁺ and Mg²⁺) and dissociated with pre-warmed StemPro Accutasse (Gibco). Cells were washed with passaging medium (KnockOut DMEM/F-12, 2 nM GlutaMAX, 1% Antibiotic/Antimycotic) in a 1:10 ratio, centrifuged at 300×g for 5 min and resuspended in passaging medium. Cells were counted via trypan blue exclusion before plating.
For proliferation, 6-well TC-Treated plastic plates were coated with Geltrex (Gibco), diluted in KnockOut DMEM/F12 (Gibco) with a final concentration of 1:120.
NSCs were proliferated in StemPro Serum Free Proliferation Medium (KnockOut DMEM/F-12, 2% StemPro Supplement, 10 ng/ml EGF, 10 ng/ml FGF2, 2 mM GlutaMAX, and 1% Antibiotic/Antimycotic). Medium was changed every other day until cells reached 100% confluency and were subsequently passaged either for further proliferation or differentiation. Cells used for all experiments came from large batches expanded and frozen at passages (P) 3, 23 and 38 to ensure consistency. Cells were always allowed to proliferate for at least two passages before being used for experiments. Plates were incubated at 37 °C with 5% CO2 in a humidified atmosphere.

For preparation of cortical neural stem cells [54 (link)], cells were plated into Nunc 90 mm petri dishes, previously coated with Cell Start (Gibco), at a seeding density of 500 x10⁵ cells/ml. Neural stem cells were cultured in StemPro NSC SFM composed of: Knockout D-MEM / F12; Glutamax (2mM); bFGF (20ng/ml); EGF (20ng/ml); StemPro Neural Supplement (2%); all from (Gibco). Under these growth conditions at 7 DIV cells were proliferative and were Nestin and Ki67 positive.