N-2 Plus Media Supplement

  (16 citations)
(2 Reviews)
Datasheet / CoA / SDS
Product Details
Assay Procedure
Citations (16)
Supplemental Products
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.



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
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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
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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.

Product Datasheets

Certificate of Analysis Lookup

To download a Certificate of Analysis, please enter a lot number in the search box below.

Note: Certificate of Analysis not available for kit components.

Certificate of Analysis Lookup

Certificate of Analysis Request Form

To download a Certificate of Analysis, please enter the catalog and lot numbers in the search box below.

Note: Certificate of Analysis not available for kit components.

Preparation and Storage
  • Stability & Storage
    Store the unopened product at -20 to -70 °C. Use a manual defrost freezer and avoid repeated freeze-thaw cycles. Do not use past expiration date.
Background: Neural Stem Cells

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.

  • Alternate Names:
    Neural Stem Cells
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


  • 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


  • Serological pipettes
  • Pipettes and pipette tips
  • 2 µm filter unit


  • 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.


R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.

16 Citations: Showing 1 - 10
Filter your results:

Sample Type
  1. Activated CD8+ T lymphocytes inhibit neural stem/progenitor cell proliferation: role of interferon-gamma.
    Authors: Hu S, Rotschafer J, Lokensgard J, Cheeran M
    PLoS ONE, 2014;9(8):e105219.  2014
  2. Proinflammatory cytokine-induced tight junction remodeling through dynamic self-assembly of claudins.
    Authors: Capaldo C, Farkas A, Hilgarth R, Krug S, Wolf M, Benedik J, Fromm M, Koval M, Parkos C, Nusrat A
    Mol Biol Cell, 2014;25(18):2710-9.  2014
  3. Isolation of cancer stem cells from three human glioblastoma cell lines: characterization of two selected clones.
    Authors: Iacopino F, Angelucci C, Piacentini R, Biamonte F, Mangiola A, Maira G, Grassi C, Sica G
    PLoS ONE, 2014;9(8):e105166.  2014
  4. miR-17 regulates the proliferation and differentiation of the neural precursor cells during mouse corticogenesis.
    Authors: Mao S, Li H, Sun Q, Zen K, Zhang C, Li L
    FEBS J, 2014;281(4):1144-58.  2014
  5. Induction of intestinal stem cells by R-spondin 1 and Slit2 augments chemoradioprotection.
    Authors: Zhou, Wei-Jie, Geng, Zhen H, Spence, Jason R, Geng, Jian-Guo
    Nature, 2013;501(7465):107-11.  2013
  6. Transcription factor-mediated reprogramming of fibroblasts to expandable, myelinogenic oligodendrocyte progenitor cells.
    Authors: Najm, Fadi J, Lager, Angela M, Zaremba, Anita, Wyatt, Krysta, Caprariello, Andrew V, Factor, Daniel C, Karl, Robert T, Maeda, Tadao, Miller, Robert H, Tesar, Paul J
    Nat Biotechnol, 2013;31(5):426-33.  2013
  7. Matrix metalloproteinase-10 is required for lung cancer stem cell maintenance, tumor initiation and metastatic potential.
    Authors: Justilien V, Regala RP, Tseng IC
    PLoS ONE, 2012;7(4):e35040.  2012
  8. Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro.
    Authors: Spence JR, Mayhew CN, Rankin SA
    Nature, 2011;470(7332):105-9.  2011
  9. Bmi1 is essential in Twist1-induced epithelial-mesenchymal transition.
    Authors: Yang MH, Hsu DS, Wang HW
    Nat. Cell Biol., 2010;12(10):982-92.  2010
  10. The giant protein AHNAK involved in morphogenesis and laminin substrate adhesion of myelinating Schwann cells.
    Authors: Salim C, Boxberg YV, Alterio J, Féréol S, Nothias F
    Glia, 2009;57(5):535-49.  2009
  11. Enhanced radiosensitivity and radiation-induced apoptosis in glioma CD133-positive cells by knockdown of SirT1 expression.
    Authors: Chang CJ, Hsu CC, Yung MC, Chen KY, Tzao C, Wu WF, Chou HY, Lee YY, Lu KH, Chiou SH, Ma HI
    Biochem. Biophys. Res. Commun., 2009;380(2):236-42.  2009
  12. Efficient Serum-Free Derivation of Oligodendrocyte Precursors from Neural Stem Cell-Enriched Cultures.
    Authors: Rao RC, Boyd J, Padmanabhan R, Chenoweth JG, McKay RD
    Stem Cells, 2008;0(0):.  2008
  13. Efficient induction of oligodendrocytes from human embryonic stem cells.
    Authors: Kang SM, Cho MS, Seo H, Yoon CJ, Oh SK, Choi YM, Kim DW
    Stem Cells, 2006;25(2):419-24.  2006
  14. Embryonic stem cell-derived neuron models of Parkinson's disease exhibit delayed neuronal death.
    Authors: Yamashita H, Nakamura T, Takahashi T, Nagano Y, Hiji M, Hirabayashi T, Amano T, Yagi T, Sakai N, Kohriyama T, Matsumoto M
    J. Neurochem., 2006;98(1):45-56.  2006
  15. Neuronal and glial expression of the adhesion molecule TAG-1 is regulated after peripheral nerve lesion or central neurodegeneration of adult nervous system.
    Authors: Soares S, Traka M, von Boxberg Y, Bouquet C, Karagogeos D, Nothias F
    Eur. J. Neurosci., 2005;21(5):1169-80.  2005
  16. High-level expression of functional chemokine receptor CXCR4 on human neural precursor cells.
    Authors: Ni HT, Hu S, Sheng WS, Olson JM, Cheeran MC, Chan AS, Lokensgard JR, Peterson PK
    Brain Res. Dev. Brain Res., 2004;152(2):159-69.  2004
Expand to show all Citations


  1. 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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