Dopaminergic lineage committed Neural Stem Cells (DA-NSCs) transitioned for differentiation into dopaminergic neurons. When thawed into Dopaminergic Differentiation Media and cultured in low oxygen conditions for 21-28 day these DA-NSCs will produce a robust population of functional neurons with 50-70% of the neurons being TH+ dopaminergic neurons as well as astrocytes and oligodentrocytes. Will last in culture for greater than 60 days.
Required Equipment:1. All procedures should be conducted inside a biological safety cabinet such as a NuAire Class II Type A2
2. The dopaminergic transitioned cells can be maintained with an antibiotic if required in order to prevent bacterial contamination. All our lines have been tested with gentamycin (30ug/ml)
3. Incubator must be low oxygen capable such as the NuAire 4950 with the following settingsa. 6% CO2b. 2-4% O2c. 95% humidityd. 37°CNote: To obtain dopaminergic neurons it is essential to culture the dopaminergic transitioned NSCs in low oxygen conditions for optimal differentiation into a robust population of dopaminergic neurons. Atmospheric oxygen incubators may not be used for differentiation of dopaminergic transitioned NSCs.
Required Supplies:1. Dopaminergic Transitioned Neural Stem Cells2. Dopaminergic Differentiation Medium includes:a. Dopaminergic Differentiation Basal Mediumb. Dopaminergic Differentiation Supplement Pack containing:Dopa Differentiation Base Supplement (contains glutamine)LamininBDNFGDNFArteminNeurtruinProprietary Factor 103. Poly-d-lysine coated plates (e.g. BD 96-well Biocoat 354640 or 384-well Biocoat 354836 or see coating method in protocol)4. Laminin/PDL coated plates (first coat with PDL then coat with Laminin)
Storage and Stability:Upon receiving, immediately transfer cells from dry ice to liquid nitrogen and keep the cells in liquid nitrogen until cell culture is needed. Stable for 6 months from the date of purchase if appropriate media and reagents are used, and the recommended protocols for storage and use are followed.
