N-2 Plus Media Supplement

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.

Components

  • 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.

Data Examples

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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