B-27™ Plus Supplement (50X) - FAQs

Yes, the protein content in this product is high enough so filtering through a low protein binding filter as a 50X or 1X in solution should not be a problem.

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No, it is not recommended to freeze media due to the potential of precipitates forming upon thaw. Inorganic salts and amino acids in the formulation may come out of solution when exposed to temperature fluctuations. These precipitates will not go back into solution easily once formed.

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After long-term storage at -20 degrees C, Human Keratinocyte Growth Supplement (HKGS) (Cat. Nos. S0015, S001K) may contain a small amount of precipitate, which is normal. This precipitate is formed from cold insoluble material in the BPE component of the HKGS and will not affect the performance of the product. A similar precipitate may be seen in the BPE component of the HKGS Kit.

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We do not recommend thawing B-27 Supplement (Cat. No. A3582801) in a water bath or incubator. We recommend thawing the B-27 Supplement at 4 degrees C overnight.

Note: Remaining B-27 Plus Supplement (50X) may be aliquoted into working volumes and stored at -20 to -5 degrees C. Do not freeze-thaw B-27 Plus Supplement (50X) more than twice. Once thawed do not leave thawed supplement at 4 degrees C for more than two weeks.

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We recommend using Neurobasal Plus Medium with B-27 Plus Supplement for long-term culture of mixed hippocampal cells. For short-term culture of a pure population of hippocampal neurons, we would recommend using Neurobasal-A Medium with B-27 Supplement.

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Yes, we do offer B-27 Supplement, minus insulin (Cat. Nos. A1895601 and A1895602). All our other B-27 supplements do contain insulin.

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We recommend using the B-27 Plus Neuronal Culture System when culturing neurons on Poly-D-Lysine. The B-27 Plus Neuronal Culture System is a complete media system that includes the B-27 Plus Supplement and the Neurobasal Plus Medium.

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Yes, we have shown improved electrophysiological activity with the B-27 Plus Neuronal Culture System

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B-27 Plus Supplement is an optimized formulation of classic B-27 Supplement with a streamlined manufacturing process. No small molecules or growth factors have been added.

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We recommend performing half medium exchanges every 2-3 days post-plating. Make sure that neurons are not completely exposed to air by not removing all of the medium during exchanges.

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We do not recommend thawing at 37 degrees C. Best results are observed upon thawing at room temperature for approximately 2 hrs or overnight at 2-8 degrees C.

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We recommend thawing it at 4 degrees C overnight or at room temperature for 2 hours. We don't recommend thawing in a water bath or incubator.

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HuC/D is an antibody specific to neuronal cell bodies. It can be used to quantitate neurons in culture.

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In primary neuronal cultures, this is typically a function of uneven coating or if neurons are plated at too high of a density. In NSC differentiation experiments, this could be due to overproliferation of neural progenitor cells. Differentiating with the B-27 Plus Neuronal Culture System and CultureOne Supplement can eliminate more than 75% of neural progenitor cells and the clumps they form. Please see the CultureOne Supplement User Guide (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0016204_CultureOneSupplement_UG.pdf) for more information.

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B-27 Plus Supplement can be stored for up to one year at -5 to -30 degrees C.
Neurobasal Plus Medium can be stored for up to one year at 2-8 degrees C.
Both products should be protected from light.

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Yes, B-27 Plus Supplement can be stored at 4 degrees C for up to 2 weeks post-thaw.

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Complete Neurobasal Plus Medium should be used within 4 weeks after supplementation with B-27 Plus Supplement.

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We recommend using B-27 Plus Supplement and Neurobasal Plus Medium instead of the regular versions of B-27 Supplement and Neurobasal Medium products (not specialty versions like minus Insulin, minus Vitamin-A or Neurobasal-A) in protocols used for:

- Maintenance of primary rat, mouse and human PSC-derived and fetal-derived neurons
- Differentiation of human PSC-derived and fetal-derived neural stem cells (NSCs) to neurons

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The B-27 Plus Neuronal Culture System was designed for culturing neuronal cells. We have not tested it in other cells.

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Add CultureOne Supplement at day 0 with the B-27 Plus Neuronal Culture System to fully suppress both astrocytes and oligodendrocytes with no detrimental effect on neurons. Delaying the addition of CultureOne Supplement to later time points results in increasing levels of astrocytes. Refer to the CultureOne Supplement User Guide (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0016204_CultureOneSupplement_UG.pdf) for more information.

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B-27 Plus Supplement and Neurobasal Plus Medium have been designed to work synergistically together for maximal performance. They can be used with other basal media or supplements if necessary.

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We have maintained primary rat cortical neurons up to 8 weeks and rat hippocampal neurons up to 4 weeks. Mouse cortical neurons were maintained for 4 weeks. Human iPSC-derived neurons that have been differentiated from neural stem cells (NSCs) for 9 days have been maintained for 5 weeks. All cells tested with the B-27 Plus System were significantly healthier than cells maintained in culture using the classic products across all time points.

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No, we do not recommend the B-27 Plus System for expanding proliferating cells. We recommend using StemPro NSC SFM, Neural Induction Medium Supplement or B-27 Supplement minus Vitamin A for growing proliferative cells, such as NSCs.

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Yes, we recommend that you aliquot when you first thaw it and thereafter subject the aliquot to no more than 2 additional freeze/thaw cycles.

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We streamlined the manufacturing process, implemented more stringent acceptance criteria and enhanced QC assays for qualification of raw materials and release of finished product to enable better lot-to-lot consistency.

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Key amino acids and buffering components have been optimized in Neurobasal Plus Medium. No small molecules or growth factors have been added.

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We offer the following B-27 supplement products, which are developed for specific applications:

- Gibco B-27 Serum-free Supplement (Cat. No. 17504044): Complete formulation for the growth of long-term viability of neurons.
- Gibco B-27 Supplement minus AO (antioxidants) (Cat. No. 10889038): Five antioxidants are removed. The supplement is ideal in studies of oxidative damage, apoptosis, or applications in which free radical damage to neurons occurs.
- Gibco B-27 Supplement minus Insulin (Cat. No. A1895601): Insulin is removed. The supplement is ideal for studies of insulin secretion and insulin receptors.
- Gibco B-27 Supplement minus Vitamin A (Cat. No. 12587010): Vitamin A which induces neural differentiation is removed. The supplement is ideal for proliferation of stem cells.

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As a general guideline, we recommend using Gibco B-27 Plus Supplement to culture neural stem cells, hippocampal, and other CNS neurons.

- For neuroblastomas, or post-mitotic neurons from both PNS and CNS, Gibco N-2 Supplement can be used.
- For primary glial cells (astrocytes) or tumor cell lines of astrocytic phenotype (astrocytes and gliomas), or oligodendrocytes, Gibco G-5 Supplement can be used.