N-2 Plus Media Supplement
R&D Systems, part of Bio-Techne | Catalog # AR003
Key Product Details
Summary for N-2 Plus Media Supplement
For culturing neural progenitor cells and their differentiated derivatives.
Key Benefits
- Optimized for NPC expansion and differentiation
- Bovine Insulin is screened for batch-to-batch performance consistency
- Chemically-defined and serum-free
- Matures and maintains a variety of NPC-differentiated cell types
Why Culture Neural Progenitor Cells (NPCs) under Fully Defined Conditions?
Serum-free, defined media are routinely used as an alternative to standard serum-containing media in order to reduce unwanted experimental variability.
Uncontrolled variables commonly associated with serum-supplemented media, such as indeterminate levels of vitamins, hormones, and growth factors are eliminated, and the potential for contamination by infectious agents is reduced when cells are cultured under defined conditions. In addition, serum-free media offers researchers the ability to specifically design the culture media for a particular experimental question.
The N-2 Plus Media Supplement:
- Contains high quality factors to support reproducible and efficient NPC expansion when combined with appropriate growth factors.
- Is fully defined to reduce unwanted experimental variation.
- Has been developed and optimized using neural cells.
N-2 Plus Media Supplement Components
Supplied as a 100X concentrate in water, this media supplement contains the following high quality factors to support neural cell culture:
- Bovine Insulin (2,500 µg/mL)
- Human Transferrin (10,000 µg/mL)
- Putrescine (1,611 µg/mL)
- Selenite (0.52 µg/mL)
- Progesterone (0.63 µg/mL)
Supplied in a volume sufficient to supplement 500 mL of media at the recommended concentration.
Precautions
This product contains human transferrin. This transferrin was purified from donor plasma and tested at the donor level using an FDA licensed method and found to be non-reactive for anti-HIV-1/2 and Hepatitis B surface antigen.
Neural stem cells provide an excellent model for research focused on neural development and neurological disorders. R&D Systems offers ready-to-use primary cortical stem cells isolated from E14.5 Sprague-Dawley rats. In addition, primary mouse cortical stem cells isolated from E14.5 CD-1 mice are available. Every lot of R&D Systems Cortical Stem Cells is validated for a high level of Nestin expression and the capacity for multi-lineage differentiation into astrocytes, neurons, and oligodendrocytes. Our cortical stem cells are tested to ensure highest quality and lot to lot consistency. Both rat and mouse Cortical Stem Cells can be optimally expanded as monolayers or neurospheres.
To complement the use of primary neural stem cells, we offer a range of supportive products, including culture media which is specifically optimized for use with neural stem cells. We offer kits to promote the in vitro proliferation of neural precursors, and kits to differentiate neural stem cells into dopaminergic neurons or oligodendrocytes. In addition, kits are available which contain panels of antibodies designed to monitor the differentiation and identification of neural precursors, astrocytes, neurons, and oligodendrocytes.
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Neural Progenitor Cells Expanded with N-2 Plus Media Supplement Express Nestin and SOX2 Neural Progenitor Cells Expanded with N-2 Plus Media Supplement Express Nestin and SOX2.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). The cells were stained with a PE-conjugated Mouse Anti-Human Nestin Monoclonal Antibody (Catalog# Catalog # IC1259P; red histogram), a PE-conjugated Mouse Anti-Human/Mouse SOX2 Monoclonal Antibody (Catalog# Catalog # IC2018P; green histogram), or a PE-conjugated Mouse IgG2AIsotype Control (Catalog# Catalog # IC003P; open histogram). Under these conditions, cells were shown to express high levels of Nestin and SOX2, two established markers of neural multipotency. |
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Verification of Neural Progenitor Cell Multipotency Following Expansion with N-2 Plus Media Supplement Verification of Neural Progenitor Cell Multipotency Following Expansion with N-2 Plus Media Supplement.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 differentiated into neurons, astrocytes, and oligodendrocytes. Markers of lineage differentiation were detected using a Mouse Anti-Human Nestin Monoclonal Antibody (Catalog # MAB1259), 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). The cells were stained for Nestin using a NorthernLights trade; (NL)493-conjugated Donkey Anti-Mouse Secondary Antibody (Catalog # NL003; green), for beta-III Tubulin using a NL557-conjugated Donkey Anti-Mouse Secondary Antibody (Catalog # NL007; red), for GFAP using NL 557-conjugated Donkey Anti-Sheep Secondary Antibody (Catalog # NL010; red), and for O4 using an Anti-Mouse IgM secondary antibody (red). The nuclei were counterstained with DAPI (blue). View our protocol for Fluorescent ICC Staining of Stem Cells on Coverslips. |
Assay Procedure
Refer to the product datasheet for complete product details.
Briefly, completed N-2 Plus-supplemented neural cell medium is prepared using the following procedure:
- Dilute the media supplement in basal media
- Store completed media
- Use within 2 weeks
Reagents provided in the N-2 Plus Media Supplement (Catalog # AR003):
- Bovine Insulin (2,500 µg/mL)
- Human Transferrin (10,000 µg/mL)
- Putrescine (1,611 µg/mL)
- Selenite (0.52 µg/mL)
- Progesterone (0.63 µg/mL)
Reagents
- DMEM/F-12 (Invitrogen®, Catalog # 12500-062) or a basal media (e.g., Neurobasal Media from Invitrogen, Catalog # 21103-029)
- Glucose
- Glutamine
- NaHCO3
- Penicillin-Streptomycin (100X)
- Deionized or distilled water
Materials
- Serological pipettes
- Pipettes and pipette tips
- 2 µm filter unit
Equipment
- 2 °C to 8 °C refrigerator
Option 1: Mix the following components with deionized or distilled water to make 500 mL of medium.
| Component | Amount |
| DMEM/F-12 | 6 g |
| Glucose | 0.775 g |
| Glutamine | 0.0365 g |
| NaHCO3 | 0.854 g |
| N-2 Plus Media Supplement | 5 mL |
Adjust the pH to 7.2. Filter the solution (2 µm filter unit), and add 5 mL of 100X sterile Penicillin-Streptomycin solution. The medium may be stored in the dark at 2 °C to 8 °C for up to 2 weeks.
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.
Invitrogen is a registered trademark of Invitrogen Corp.