B-27® 添加剂是无血清添加剂,用于海马神经元和其他中枢神经系统(CNS)神经元的生长和保持其短期或长期活性。B-27® 添加剂是50X 的液体,可以与Neurobasal® 培养基或Neurobasal®-A 培养基组合使用,用于神经细胞培养而不需要再添加饲养层细胞。
B-27® 添加剂适和应用于大多数神经细胞培养。对于特别的应用,我们提供
其他配方的B-27® 添加剂.
质量控制检测
B-27® 添加剂 进行了18天妊娠的原代大鼠(Sprague Dawley) 的胚胎海马神经元生长检测。通过了细菌和真菌的检测. B-27® 也做了1X 浓度的内毒素水平测试。
仅供研究使用。除非另有说明,不得用于任何动物或人类治疗或诊断。
B-27®与N-2添加剂产品列表:
17504044 B-27® Supplement (50X), serum free 10 mL CNY 1371.00
12587010 B-27® Supplement (50X), minus vitamin A 10 mL CNY 1,368.00
10889038 B-27® Supplement (50X), minus antioxidants 10 mL CNY 1,366.00
A1486701 CTS™ B-27® Supplement, XenoFree 10 mL CNY 2,255.00
0080085SA B-27® Supplement (50X), custom 10 mL CNY 1,202.00
A1413701 B-27® Electrophysiology Kit 1 kit CNY 2,025.00
17502048 N-2 Supplement (100X) 5 mL CNY 956.00
A1370701 CTS™ (Cell Therapy Systems) N-2 Supplement 5 mL CNY 1,210.00
B-27 Supplement 50X
CAUTION: Human origin materials are non-reactive (donor level)
for anti-HIV 1 & 2, anti-HCV, and HBsAg. Handle in
accordance with established bio-safety practices.
Cat. No. 17504 10 mL
Intended Use
B-27 Supplement is a 50X serum-free supplement designed for the long-term
viability of hippocampal and other neurons of the central nervous system
(CNS). It is intended for laboratory research use only.
Storage Conditions: -5 to -20°C, protect from light.
Features and Benefits
• B-27 when used as a supplement to NEUROBASAL (Cat. No. 21103) has
been demonstrated to give optimal growth and long-term survival of rat
embryonic hippocampal neurons, and growth and survival of neurons
from embryonic rat striatum, substantia nigra, septum and cortex, and
neonatal rat cerebellum and dentate gyrus1.
• B-27 when used as a supplement to NEUROBASAL is effective for the
growth of tumor cell lines of neuronal origin.
• B-27 when used as a supplement to NEUROBASAL-A (Cat. No. 10888) has
been demonstrated to allow for the growth of postnatal and adult rat
hippocampal and cortical neurons after further supplementation with β-
FGF (Cat. No. 13256)2.
• B-27 Supplement has been demonstrated to allow the expansion of EGFresponsive
precursor cells from embryonic rat striatum and
mesencephalon3.
• B-27 when used as a supplement to NEUROBASAL supports the growth of
nearly pure populations of neural cells without the need of an astrocyte
feeder layer.
• B-27 Supplement contains a cocktail of antioxidants to reduce reactive
oxygen damage.
• B-27 has a one year shelf-life when stored between -5 to -200C.
Background
B-27 is an optimized serum substitute developed for low density plating and
long-term viability and growth of hippocampal and other CNS neurons. By
supplementing NEUROBASAL with B-27 and 0.5 mM L-glutamine Cat. No.
25030); excellent long-term viability of rat embryonic hippocampal neurons has
been achieved even after four weeks 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 is reduced to less than 0.5% for a nearly pure
neuronal population1.
When using B-27 as a supplement to NEUROBASAL it is suggested that 25 μM
(3.7 μg/mL) L-glutamic acid be added to the medium for the initial plating of
primary hippocampal neurons. Subsequent medium changes after day 4
should be made without glutamate. With neuroblastomas, the glutamate
should be included in the medium for both plating and subsequent media
changes.
Improved long-term survival of hippocampal neurons may be obtained by the
addition of 2-mercaptoethanol (Cat. No. 21985) at 25 μM4,5.
Application
In addition to low density growth of fetal hippocampal neurons, the combination
of B-27 and NEUROBASAL has been shown to support the growth of neurons
from embryonic rat striatium, substantia nigra, septum, cortex, and neonatal
dentate gyrus and cerebellum1.
The combination of B-27 Supplement with NEUROBASAL-A has been
demonstrated to support the growth of postnatal and adult rat hippocampal and
cortical neurons2.
The combination of B-27 Supplement with a DMEM:F12 mixture has been
demonstrated to support the expansion of EGF-responsive precursor cells from
rat embryonic striatum and mesencephalon3.
Quality Control Testing
B-27 Supplement is tested in a growth assay utilizing primary rat (Sprague
Dawley) embryonic hippocampal neurons, 18 day gestation. Additional
standard evaluations are for the absence of bacterial and fungal contaminants.
B-27 is also tested for endotoxin at a 1X concentration.
Instructions for Use
As the B-27 Supplement is supplied as a 50X concentrate, you should add 2.0
mL to 100 mL of NEUROBASAL.
The following procedures have been found effective with 18-day gestation rat
hippocampi and with neuroblastoma cell lines.
1. Coat culture vessels with a 0.05 mg/mL solution of cold poly-D-lysine (MW
30,000 - 70,000) and incubate for 1 hour or overnight. For primary cultures
use 0.15 mL/cm2 surface area. When using neuroblastoma cell lines coat
the dish with 0.04 mL/cm2 of poly-D-lysine. Poly-D-lysine solutions are
stored at -20oC in polycarbonate tubes. Poly-D-lysine should be
prescreened for toxicity.
2. Wash vessels with sterile, deionized cell culture grade water. Note:
Vessels can now be used or stored for up to 2 weeks at 4o to 10oC in
sterile deionized, distilled water. If vessels are to be stored, remove water
about 1 hour prior to use.
3. To NEUROBASAL medium, add 0.5 mM L-glutamine, 25 μM L-glutamic acid,
and 2% B-27 Supplement.
4. For primary hippocampal neurons (i.e. from Sprague Dawley rats at 18
days gestation) and other fetal neurons.
a. Embryos are recovered by C-section under nembutal anesthetic and
desired region dissected.
b. Individual cells are isolated by trituration 10 times in 1 mL Hanks' Balanced Salt
Solution w/o Ca++ and Mg++ (Cat. No. 14175) and supplemented with 1.0 mM
sodium pyruvate (Cat. No. 11360) and 10 mM HEPES (Cat. No. 15630), pH 7.4
using a 9 inch siliconized Pasteur pipette with the tip barely fire polished.
c. Divalent cations are restored by dilution with 2 volumes HBSS (Cat. No.
14025) supplemented as above.
d. After allowing non-dispersed tissues to settle for 3 min., the supernatant is
transferred to a 15 mL tube and centrifuged for 1 min. at 200 g.
e. The pellet is gently resuspended in 1 mL HBSS per brain and an aliquot
taken for counting.
f. Cells are added to the wells with supplemented NEUROBASAL at 160/mm2
or other desired concentrations.
g. Cultures maintained longer than 4 days should have one-half of the
medium changed to B-27/ NEUROBASAL without L-glutamic acid on day 4
and then once per week. If the initial culture density is higher than 640
cell/mm2, the medium should be changed twice a week.
1. Cell Lines
a. Some cell lines may require an initial attachment in 2% serumsupplemented
NEUROBASAL medium. Serum-free NEUROBASAL
supplemented with B-27 can then be added after incubation for 2 hours or
overnight.
b. To transfer cells:
Remove spent media and wash cells with HBSS (Cat. No. 14175). Detach
cells from the substratum with 0.25 % trypsin/1.0 mM EDTA Cat. No.
25300). Aspirate excess trypsin/EDTA solution. A strong tap to the vessel
after 2-4 minutes should remove cells. Dilute cells in HBSS (Cat. No.
14025) containing 0.05% soybean trypsin inhibitor (Cat. No. 17075).
Centrifuge at 1000X g for 2 min. at room temperature. Resuspend pellet in
the plating medium at the desired plating concentration.
References:
1. Brewer, G.J., Torricelli, J.R., Evege, E.K., Price, P.J. Optimized Survival of Hippocampal
Neurons in B-27 Supplemented NEUROBASALTM. A New Serum-free Medium Combination.
J. Neurosci. Res. 35:567-576 (1993).
2. Brewer, G.J. Isolation and Culture of Adult Rat Hippocampal Neurons. J. Neurosci. Methods
71: 145-158 (1997).
3. Svendsen, C.N., Fawcett, J.W., Bentlage, C., Dunnett, S.B. Increased Survival of Rat EGFGenerated
CNS Precursor Cells using B-27 Supplemented Medium Exp. Brain Res. 102:
407-414 (1995).
4. Grill, R.J., Jr., Pixley, S.K. 2-Mercaptoethanol Is A Survival Factor For Olfactory, Cortical
and Hippocampal Neurons In Short-term Dissociated Cell Culture. Brain Res. 613:168-172
(1993).
5. Ishii, K., Katayama, M., Hori,K., Yodoi, J., Nakanishi, T. Effects of 2-Mercaptoethanol on
Survival and Differentiation of Fetal Mouse Brain Neurons Cultured In Vitro. Neurosci.
Letters 163: 159-162 (1993).
For further information on this or other GIBCOTM products, contact Technical
Services at the following:
United States TECH-LINE SM : 1 800 955 6288
Canada TECH-LINE: 1 800 757 8257
Outside the U.S. and Canada, refer to the GIBCO products catalogue for the
TECH-LINE in your region.
You may also contact your Invitrogen Sales Representative or our World Wide
Web site at www.invitrogen.com.
For research use only.
CAUTION: Not intended for human or animal
diagnostic or therapeutic uses.
June 2001 Form No. 3889