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N-2 Plus Media Supplement Summary
Kit Summary
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 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. As no testing can offer complete assurance of freedom from infectious agents, these reagents should be handled as if capable of transmitting infection.
Data Examples
 | 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#IC1259P; red histogram), a PE-conjugated Mouse Anti-Human/Mouse SOX2 Monoclonal Antibody (Catalog#IC2018P; green histogram), or a PE-conjugated Mouse IgG2AIsotype Control (Catalog#IC003P; open histogram). Under these conditions, cells were shown to express high levels of Nestin and SOX2, two established markers of neural multipotency. |
 | 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. |
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
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)
Other Supplies Required
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
Procedure Overview
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.
Citations (21)
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Citations are publications that use Bio-techne products. Selected citations for N-2 Plus Media Supplement include:
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Species: Mouse
Sample Types: Whole Cells
T Kobayashi et al. (2019), Enhanced lysosomal degradation maintains the quiescent state of neural stem cells Nat Commun, 10(1):5446.
PMID: 31784517 -
Species: Rat
Sample Types: Whole Cells
Applications: Cell CultureJM Yi et al. (2019), Ameliorative effects of aqueous extract of Forsythiae suspensa fruits on oxaliplatin-induced neurotoxicity in vitro and in vivo BMC Complement Altern Med, 19(1):339.
PMID: 31783843 -
Species: Mouse
Sample Types: Whole Cells
J Wang et al. (2018), Zika virus infected primary microglia impairs NPCs proliferation and differentiation Biochem. Biophys. Res. Commun., 0(0):.
PMID: 29453985 -
Species: Mouse
Sample Types: Whole Tissue
Applications: BioassayJJ Brown et al. (2018), Reovirus-Induced Apoptosis in the Intestine Limits Establishment of Enteric Infection J. Virol., 0(0):.
PMID: 29514905 -
Species: Mouse
Sample Types: Whole Cells
Applications: BioassayN Boukelmoun et al. (2018), Mitochondrial transfer from mesenchymal stem cells to neural stem cells protects against the neurotoxic effects of cisplatin Acta Neuropathol Commun, 6(1):139.
PMID: 30541620 -
Species: Mouse
Sample Types: Whole Cells
Mao S et al. (2014), miR-17 regulates the proliferation and differentiation of the neural precursor cells during mouse corticogenesis. FEBS J, 281(4):1144-58.
PMID: 24314167 -
Species: Human
Sample Types: Whole Cells
Iacopino F et al. (2014), Isolation of cancer stem cells from three human glioblastoma cell lines: characterization of two selected clones. PLoS ONE, 9(8):e105166.
PMID: 25121761 -
Species: Mouse
Sample Types: Whole Cells
Capaldo C et al. (2014), Proinflammatory cytokine-induced tight junction remodeling through dynamic self-assembly of claudins. Mol Biol Cell, 25(18):2710-9.
PMID: 25031428 -
Species: Mouse
Sample Types: Whole Tissue
Hu S et al. (2014), Activated CD8+ T lymphocytes inhibit neural stem/progenitor cell proliferation: role of interferon-gamma. PLoS ONE, 9(8):e105219.
PMID: 25133679 -
Species: Mouse
Sample Types: Whole Cells
Applications: BioassayNajm et al. (2013), Transcription factor-mediated reprogramming of fibroblasts to expandable, myelinogenic oligodendrocyte progenitor cells. Nat Biotechnol, 31(5):426-33.
PMID: 23584611 -
Species: Mouse
Sample Types: Whole Cells
Zhou et al. (2013), Induction of intestinal stem cells by R-spondin 1 and Slit2 augments chemoradioprotection. Nature, 501(7465):107-11.
PMID: 23903657 -
Species: Mouse
Sample Types: Whole Cells
Applications: BioassayJustilien V et al. (2012), Matrix metalloproteinase-10 is required for lung cancer stem cell maintenance, tumor initiation and metastatic potential. PLoS ONE, 7(4):e35040.
PMID: 22545096 -
Species: Human
Sample Types: Whole Cells
Applications: BioassaySpence JR et al. (2011), Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro. Nature, 470(7332):105-9.
PMID: 21151107 -
Species: Human
Sample Types: Whole Cells
Yang MH et al. (2010), Bmi1 is essential in Twist1-induced epithelial-mesenchymal transition. Nat. Cell Biol., 12(10):982-92.
PMID: 20818389 -
Species: Human
Sample Types: Whole Cells
Applications: BioassayChang CJ et al. (2009), Enhanced radiosensitivity and radiation-induced apoptosis in glioma CD133-positive cells by knockdown of SirT1 expression. Biochem. Biophys. Res. Commun., 380(2):236-42.
PMID: 19166820 -
Species: Human
Sample Types: Whole Cells
Salim C et al. (2009), The giant protein AHNAK involved in morphogenesis and laminin substrate adhesion of myelinating Schwann cells. Glia, 57(5):535-49.
PMID: 18837049 -
Species: Mouse
Sample Types: Whole Cells
Applications: BioassayRao RC et al. (2008), Efficient Serum-Free Derivation of Oligodendrocyte Precursors from Neural Stem Cell-Enriched Cultures. Stem Cells, 0(0):.
PMID: 18403757 -
Species: Human
Sample Types: Whole Cells
Applications: BioassayKang SM et al. (2006), Efficient induction of oligodendrocytes from human embryonic stem cells. Stem Cells, 25(2):419-24.
PMID: 17053214 -
Species: Mouse
Sample Types: Whole Cells
Yamashita H et al. (2006), Embryonic stem cell-derived neuron models of Parkinson's disease exhibit delayed neuronal death. J. Neurochem., 98(1):45-56.
PMID: 16805795 -
Species: Rat
Sample Types: Whole Cells
Soares S et al. (2005), Neuronal and glial expression of the adhesion molecule TAG-1 is regulated after peripheral nerve lesion or central neurodegeneration of adult nervous system. Eur. J. Neurosci., 21(5):1169-80.
PMID: 15813926 -
Species: Human
Sample Types: Whole Cells
Ni HT et al. (2004), High-level expression of functional chemokine receptor CXCR4 on human neural precursor cells. Brain Res. Dev. Brain Res., 152(2):159-69.
PMID: 15351504
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N-2 Plus Media Supplement
Verified Customer | Posted 04/04/2018 -
N-2 Plus Media Supplement
Verified Customer | Posted 12/01/2017Great growth and stability of E14 Neuronal stem cells
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FAQs
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What is the difference between N-2 Plus Media Supplement (Catalog # AR003) and N-2 MAX Media Supplement (Catalog # AR009)?
The N-2 Plus Media Supplement (Catalog # AR003) contains Bovine Insulin whereas N-2 MAX Media Supplement (Catalog # AR009) contains Recombinant Human Insulin. All other media components and concentrations in N-2 Plus and N-2 MAX are the same. These two products perform comparably in side-by-side testing. Due to a limited supply of bovine insulin, the products are priced differently.
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Can the N-2 MAX Media Supplement be thawed, aliquoted and refrozen to be used with smaller batches of basal media?
Yes, this will not affect the stability of the supplement. Limited freeze-thaw of the supplement to use it in small batches is acceptable.
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Can the N-2 Plus Media Supplement be thawed, aliquoted and refrozen to be used with smaller batches of basal media?
Yes, this will not affect the stability of the supplement. Limited freeze-thaw of the supplement to use it in small batches is acceptable.
