A serum-free, chemically defined, concentrated media supplement formulated to provide optimal growth conditions for neural stem cell expansion (Catalog # AR003). N-2 Plus is composed of Bovine Insulin, Human Transferrin, Putrescine, Selenite, and Progesterone. The supplement is supplied as a 100X concentrate in water.
- Bovine Insulin (2500 μg/mL)
- Human Transferrin (10,000 μg/mL)
- Putrescine (1611 μg/mL)
- Selenite (0.52 μg/mL)
- Progesterone (0.63 μg/mL)
N-2 Plus Medium Preparation
Option 1: Mix the following components with deionized or distilled water to make 500 mL of medium. Adjust the pH to 7.2. Filter the solution (2 μm filter unit), and add 5 mL of 100X sterile Penicillin-Streptomycin solution (Invitrogen®, Catalog # 15140-148). The medium may be stored in the dark at 2 °C to 8 °C for up to 2 weeks.
- DMEM/F-12 (Invitrogen, Catalog # 12500-062) 6 g
- Glucose (Sigma, Catalog # G6152) 0.775 g
- Glutamine (Sigma, Catalog # G8540) 0.0365 g
- NaHCO3 (Sigma, Catalog # S5761) 0.845 g
- N-2 Plus Media Supplement (100X) 5 mL
Option 2: Dilute 100-fold with a basal media (e.g. Neurobasal Media from Invitrogen, Catalog # 21103-029) before use. The medium may be stored in the dark at 2 °C to 8 °C for up to 2 weeks.
||Detection of Nestin and SOX2 in Neural Progenitor Cells by Flow Cytometry. Human neural progenitor cells were cultured for 7 days in media supplemented with 1X N-2 Plus Media Supplement (Catalog # AR003) and 20 ng/mL of Recombinant Human FGF basic (Catalog # 233-FB). Cells were stained with a PE-conjugated Mouse Anti-Human Nestin Monoclonal Antibody (Catalog # IC1259P, red histogram), a PE-conjugated Mouse Anti-Human/Mouse SOX2 Monoclonal Antibody (Catalog # IC2018P, green histogram), or a PE-conjugated Mouse IgG2A Isotype Control (Catalog # IC003P, filled histogram). Under these conditions, cells were shown to express high levels of Nestin and SOX2, two established markers of neural multipotency.
|Lineage Differentiation of Neural Progenitor Cells. Human neural progenitor cells were cultured for 7 days in media supplemented with 1X N-2 Plus Media Supplement (Catalog # AR003) and 20 ng/mL of Recombinant Human FGF basic (Catalog # 233-FB). Following the withdrawal of FGF basic, neural progenitor cells randomly differentiate into neurons, astrocytes, and oligodendrocytes. Markers of lineage differentiation were detected using a Mouse Anti-Human Nestin Monoclonal Antibody (Catalog # MAB159), a Mouse Anti-Neuron-specific beta-III Tubulin (clone TuJ-1) Monoclonal Antibody (Catalog # MAB1195), a Sheep Anti-Human GFAP Antigen Affinity-purified Polyclonal Antibody (Catalog # AF2594), and a Mouse Anti-Oligodendrocyte Marker O4 Monoclonal Antibody (Catalog # MAB1326). Cells were stained for Nestin using a NorthernLights™ 493-conjugated Donkey Anti-Mouse Secondary Antibody (Catalog # NL003; green), for beta-III Tubulin using a NorthernLights™ 557-conjugated Donkey Anti-Mouse Secondary Antibody (Catalog # NL007; red), for GFAP using NorthernLights™ 557-conjugated Donkey Anti-Sheep Secondary Antibody (Catalog # NL010; red), and for O4 using an Anti-Mouse IgM secondary antibody (red).
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