Cat. no. 21103, 12348, 10888, 12349, neurobasal (tm) and neurobasal (tm)-a media, insert no. 3946

NEUROBASAL and NEUROBASAL -A must be supplemented with N2 orB27 and 0.5 mM L-glutamine. For the initial plating of embryonic primaryhippocampal neurons, it is suggested that 25 µM (3.7 µg/mL) glutamatebe added to the NEUROBASAL medium. Both media support the growthof nearly pure populations of neural cells without the need of an astrocytefeeder layer. Both media, when supplemented with B27, contain anti- EUROBASAL
and NEUROBASAL
oxidants to reduce reactive oxygen damage and they do not contain theexcitatory amino acids, glutamate and aspartate, making it amenable to thestudy of these neurotransmitters.
Serum-free basal medium for the long-term viability of hippocampal and other neurons of the CNS. Cell Plating:
a. Autoclaved glass coverslips (12 mm diameter Assistant brand from
Carolina Biological) previously coated with 50 µg/mL poly-D-lysine in water (135 kD, Sigma) should be applied overnight, aspirated, rinsed once with water and allowed to dry one hour.
b. Cells are plated at the desired concentration (i.e. 90-320 cells/mm2) in NEUROBASALTM
60 - 150µL NEUROBASAL -A/B27(postnatal) or NEUROBASAL NEUROBASALTM
c. One hour after plating and incubation (ambient oxygen with 5 % CO2 is Medium without
Phenol Red
quickly picked up, allowed to drain and transferred to 0.4 mL NEUROBASALTMA
NEUROBASAL -A/B27 in a 24-well plate at 370 C. For postnatal neurons, the medium is aspirated, the coverslips rinsed once with NEUROBASALTMA
warm NEUROBASAL -A and the cells refed with NEUROBASAL -A Medium without
supplemented with B27, 0.5 mM L-glutamine, 1% penicillin- Phenol Red
streptomycin (Cat. No. 15070) and 5 ng/mL β-FGF (Cat. No. 13256).
d. When culturing the cells for longer than 4 days, one-half of the medium is removed on day 3 or 4 and replaced with an equal volume ofmedium now containing 10 ng/mL β-FGF (postnatal neurons).
Prior to use, NEUROBASAL media must be supplemented with 0.5 mM L-glutamine for primary cells and 2.0 mM L-glutamine for neuronal Subsequent medium changes for prenatal neurons (4 days) should be phenotype tumor cells or neural stem cells. In addition, either a serum- made with NEUROBASAL without glutamate, to reduce glutamate free supplement or serum needs to be added. Recommended supplement toxicity in the culture. With neuroblastomas, the glutamate should be is B-27 Supplement (Cat. No. 17504) for hippocampal and other CNS included in the medium for both plating and subsequent media neurons. For embryonic neurons the addition of 25 uM glutamic acid is also recommended for the plating step. For neural stem cells N-2 Improved long-term survival of hippocampal neurons may be obtained by Supplement (Cat. No. 17502) or B-27 Supplement without retinoic acid(Cat. No. 12587) is recommended. For tumor cell lines of glial origin G-5 the addition of 2-mercaptoethanol (Cat. No. 21985) at 25 µM4.
Protocols for isolation of primary hippocampal and cortical
neurons can be found in the Tech-online portion of the Invitrogen
NEUROBASAL - formulations are tested for pH, osmolality, endotoxin and website, www.invitrogen.com
the absence of bacterial and fungal contamination. For growth promotionand absence of toxicity, the medium is supplemented with B27 and tested Units of 500mL
in a growth assay utilizing a B104 (neuroblastoma) cell line Storage conditions: 2o to 8o C, in the dark
References
1 Brewer, G.J. Isolation and culture of adult rat hippocampal neurons. J Neurosci.
Intended Use
Methods: in press 71: 145-158 (1997).
NEUROBASAL Cat. No. 21103 - when supplemented with B27 is intended to give Brewer, G.J., Torricelli, J.R., Evege, E.K. and Price,P.J. Optimized survival of optimal growth and long- term survival of rat embryonic hippocampal neurons1, hippocampal neurons in B27 supplemented NEUROBASAL. A new serum- and growth and survival of neurons from embryonic rat striatum, substantia free medium combination. J. Neurosci. Res. 35: 567-576 (1993).
nigra, septum and cortex, and neonatal rat cerebellum and dentate gyrus2.
Brewer, G.J. Serum-free B27/Neurobasal medium supports differential growth NEUROBASAL Cat. No. 12348 - Specific use for receptor studies such as of neurons from the striatum, substantia nigra, septum, cerebral cortex, estrogenic receptors, downstream protein purification studies or other processes cerebellum, and dentate gyrus. J. Neurosci. Res. 42, 674-683 (1995).
where the presence of Phenol Red is undesirable Price, P.J. and Brewer, G.J. Serum-free mediua for neural cell culture.
Protocols for Neural Cell Culture, 3rd edition. Chapter19. Eds. S. Federaff and EUROBASAL -A Cat. No. 10888 - when supplemented with B272 and β-FGF is A. Richardson, Humana Press 255-264, (2001).
intended to maintain long-term growth and viability of rat postnatal and adult 3 Bottenstein, J.E. Defined Media For Dissociated Neural Cultures. In: Current Methods In Cellular Neurobiology 4:107-130 (J.L. Barker, ed.), John Wiley
-The osmolality of NEUROBASAL -A was demonstrated to be optimal for 4 Grill, R.J., Jr., Pixley, S.K. 2-Mercaptoethanol Is A Survival Factor For - NEUROBASAL when supplemented with N23, B27 or serum is effective for the Olfactory, Cortical and Hippocampal Neurons In Short-term Dissociated Cell growth of tumor cell lines of neuronal origin.
Culture. Brain Res. 613:168-172 (1993).Ishii, K., Katayama, M., Hori, K.,
NEUROBASAL -A Cat. No. 12349 - Specific use for receptor studies such as
Yodoi, J., Nakanishi, T. Effects of 2- Mercaptoethanol on Survival and estrogenic receptors, downstream protein purification studies or other processes Differentiation of Fetal Mouse Brain Neurons Cultured In Vitro. Neurosci. where the presence of Phenol Red is undesirable Letters 163: 159-162 (1993).
Background
For further information on this or other GIBCO® products, contactTechnical Services at the following: NEUROBASAL with B27 (Cat. No. 17504) has shown excellent long-termviability of rat embryonic hippocampal neurons even after four (4) weeks United States TECH-LINE SM : 1 800 955 6288 in culture with greater than 90% viability for cells plated at 640/mm2 and greater than 50% viability for cells plated at 160/mm2. Glial cell growth at Outside the U.S. and Canada, refer to the GIBCO products catalogue for five (5) days is reduced to less than 0.5% for a nearly pure neuronal population1.
The growth of adult CNS neurons requires gentle separation of their You may also contact your Invitrogen Sales Representative or our World numerous connections, a density gradient for the separation of Wide Web site at www.invitrogen.com.
oligodendrocytes and enrichment of neurons, an adequate substrate forattachment and a dedicated medium for growth. Brewer1 has shown that For research use only.
NEUROBASAL -A supplemented with B27 and adequate isolation methods CAUTION: Not intended for human or animal
permit the isolation of spherical remnants of hippocampal neurons from diagnostic or therapeutic uses.
any age rat and promote the regeneration of axon and dendrite-likeprocesses.
September 2006
Form No. 3946

Protocols for isolation of primary hippocampal and cortical neurons can be found in the Tech-online portion of the Invitrogen website, www.invitrogen.com

CAUTION: Not intended for human or animal

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