Why Culture Neurons in Serum-Free N21-MAX Media Supplement?
The N21-MAX Media Supplement improves upon the traditional B271 and NS212 neuronal supplements, offering a serum-free and fully-defined formulation that is optimized for the reliable maturation, consistent health, and superior function of neurons in culture. There is an increase in the use of B27-like supplements to optimize stem cell and 3D cell culture media. N21-MAX improves stem cell differentiation and increases the viability and health of differentiated cell types during long-term cell culture conditions.
N21-MAX was designed to eliminate uncontrolled variables, such as those found in both serum-containing media and in some commercial serum-free neuronal supplements. Use of undefined media supplements can confound neuronal growth and maturation. N21-MAX contains a cocktail of factors optimized for neuronal survival, neurite extension, and synaptic maturation. Each lot of N21-MAX is tested on primary hippocampal neurons to ensure consistent performance for the user.
The N21-MAX Media Supplement:
Is fully defined and quality tested to reduce unwanted experimental variation.
Contains factors qualified to support reproducible and long-term neuron culture.
Modified from the published B27 formulation1.
Does not require an astrocyte feeder layer.
Has been tested to support the viability and growth of E18 rat hippocampal neurons.
Brewer et al., (1993) J. Neurosci. Res. 32:567
Chen et al., (2008) J. Neurosci. Methods 171:239
N21-MAX is optimized for the maturation and long-term culturing of neuronal cells. The supplement is supplied as a 50X concentrated solution and contains the following 21 components:
Supplied in a volume sufficient to supplement 500 mL of media at the recommended concentration. N21-MAX is tested for use in Neurobasal Media.
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.
N21-MAX Media Supplement Enhances Synaptic Development. E18 rat hippocampal neurons were grown for 19 days in vitro in media supplemented with either N21-MAX Media Supplement or the neural media supplement from the most widely-used competitor. Cells were incubated with the synaptic vesicle dye, SynaptoRed™ C2 (10 µM; Catalog # 5118), for 1 minute prior to depolarization with 50 mM KCl. Cells were imaged for SynaptoRed™ C2-positive synaptic puncta using the Operetta CLS™ High Content Analysis System. (A) Quantification of SynaptoRed™ C2-positive puncta shows that neurons grown in N21-MAX have an increased number of synaptic puncta compared to the competitor media. (B) Quantification of dye intensity shows that neurons grown in N21-MAX have more robust synaptic activity than neurons cultured in the competitor media. (C) Representative images of SynaptoRed™-C2 staining in neurons cultured in N21-MAX or competitor media. (D) Representative images showing the quantification of synaptic puncta in neurons cultured in N21-MAX or competitor media. Colored circles indicate a SynaptoRed™-positive synaptic puncta.
N21-MAX Reduces Spontaneous Stress Rod Formation. E18 rat hippocampal neuron cultures were plated on poly-D-lysine coated coverslips and grown for 5 days in media supplement with either N21-MAX (Catalog # AR008) or another commercial neural media supplement (Competitor). Cells were fixed and immunostained for rod formation, and indicator of cellular stress, using an antibody against cofilin (% of Neurons with Rods). A lower percentage of hippocampal neurons that were cultured in N21-MAX had spontaneous cofilin rod formation (4.5%) compared to neurons cultured in competitor media (16.8%). Data courtesy of the laboratory of Dr. James Bamburg at Colorado State University.
Improved Resolution of Rod Induction in Neurons Cultured in N21-MAX. E18 rat hippocampal neuron cultures were plated on poly-D-lysine coated coverslips and grown for 5 days in N21-MAX (Catalog # AR008). Neurons were then treated with rod-inducing agents or maintained in N21-MAX alone (Untreated). Neurons were fixed and immunostained for rod formation using an antibody against cofilin (% of Neurons with Rods). The low background of rod formation in untreated cultures enabled detection of mild rod induction using Synthetic Amyloid B, Amyloid β dimer/trimer, and TNF-α. As a positive control, treatment with an ATP depletion solution or glutamate resulted in robust rod induction. Data courtesy of the laboratory of Dr. James Bamburg at Colorado State University.
Synaptotagmin and CAM Kinase II Expression in Neurons Cultured in Media Supplemented with N21-MAX. E18 rat hippocampal neurons were grown for 21 days in vitro in media supplemented with N21-MAX Media Supplement (Catalog # AR008). Cells were stained with either (A) a Mouse Anti-Rat Synaptotagmin-1 Monoclonal Antibody (Catalog # MAB4364), followed by the NorthernLights™ (NL)557-conjugated Donkey Anti-Mouse IgG Secondary Antibody (Catalog # NL007; yellow), or (B) a Mouse Anti-Human CaM Kinase II alpha Monoclonal Antibody (Catalog # MAB5584), followed by the NL557-conjugated Donkey Anti-Mouse IgG Secondary Antibody (Catalog # NL007; green). The nuclei were counterstained with DAPI (blue). View our protocol for Fluorescent ICC Staining of Cells on Coverslips.
Increased Synaptic Puncta and Neurite Outgrowth of Primary Neurons Cultured in N21-MAX. E18 rat hippocampal neurons were grown for 21 days in vitro in media supplemented with either N21-MAX Media Supplement (Catalog # AR008) or the neural media supplement from the most widely-used competitor. Staining for Synaptotagmin (yellow) showed more robust synaptic puncta and increased neurite outgrowth in neurons cultured in N21-MAX compared to those cultured in competitor media. Cells were staineed with a Mouse Anti-Rat Synaptotagmin-1 Monoclonal Antibody (Catalog # MAB4364) followed by the NorthernLights™ (NL)557-conjugated Donkey Anti-Mouse IgG Secondary Antibody (Catalog #& NL007). Nuclei were counterstained with DAPI (blue).
N21-MAX Media Supplement Increases Efficiency of Pancreatic Cell Differentiation. Differentiation of PDX-1+ pancreatic cells from induced pluripotent stem cells (iPS2) was performed with base media containing either the N21-MAX Media Supplement (green) or the competitor equivalent (red). A) Compared to competitor media, N21-MAX improved pancreatic cell differentiation as observed by flow cytometry. B) Bar graph quantifying PDX-1+ cells in N21-MAX and the competitor media.
Store the unopened product at -20 to -70 degreesC. 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.
Neural Stem Cells
Refer to the product datasheet for complete product details.
Briefly, completed N21-MAX-supplemented 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 N21-MAX Media Supplement (Catalog # AR008):
Other Supplies Required
Basal media (e.g., Neurobasal Media from Invitrogen®, Catalog # 21103-029 or equivalent)
Pipettes and pipette tips
2 °C to 8 °C refrigerator
Dilute 50-fold with a basal media (e.g., Neurobasal Media from Invitrogen, Catalog # 21103-029) and supplement with 0.5 mM L-glutamine 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.
What advantages does the N21-MAX Media Supplement (Catalog # AR008) have over other neuronal stem cell supplements?
We have performed comparison testing in-house and have found that in long term cultures (21 days), we observe better synapse marker expression.
Additionally, one major formulation difference is that our N21 contains holo-transferrin while other supplements typically contain apo-transferrin. It is reported in the literature that holo-transferrin is preferred for neuronal cell culture. We have also tested several BSA sources to determine which is optimal for neuronal cultures.
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Other Experimental Details
Adult human stem cells showed fast growth and took less time to differentiate into neurons. Data is communicated so cannot upload image.
Other Experimental Details
Other Experimental Details
I have previously used Gibco B27®, and Gem21 NeuroPlex™ for culturing of neural cells and neuronal lines. I use typical for co- culture of neurons with astrocytes and microglia. I tried your sample N21 max from R&D and it worked very well with my neuronal cells co cultures. All of these Gibco B27®, and Gem21 NeuroPlex™ and N21 max are Supplied as a 50X concentrate and are list priced at $79 for 10 ml. I would use them all interchangeably as they all worked equally well in my co culture system.