B27

B27是在神经元细胞培养中所用的添加物,能够维持神经元细胞长期体外培养,因为神经元培养过程中不能使用血清所以用其代替,是专用于培养神经细胞的血清替代物,B27分装,是1ml、1ml 的分装然后放在-20℃冰箱里,分装完后可直接配成50ml的培养基。 B27配方 B27和N2两者只能选其一,因为B27成分是在N2基础上添加了一些成分,两者都用没有必要,只N2或B27就足够了。建议头二周先以B27成球,然后更换N2长期培养.因B27较N2有更多的添加成分,更利于细胞从打击中恢复. N2 Supplement-A: 2.5mg/mL rh Insulin 10 mg/mL Human Transferrin (Iron-Saturated) 0.52 mg/mL Sodium Selenite 1.61 mg/mL Putrescine 0.63 mg/mL Progesterone in Phosphate Buffered Saline N2 Supplement-B: 2.5 mg/mL rh Insulin 10 mg/mL Human Transferrin (Iron-Poor) 0.52 mg/mL Sodium Selenite 1.61 mg/mL Putrescine 0.63 mg/mL Progesterone in Phosphate Buffered Saline B27组成成份比较多,但是说明书上好像也没有具体提供,我在一篇文献上看到,B27的成份与该文作者自己配制高度接近: L-alanine (Sigma,cat. no. A-7627) 2 mg/ ml, biotin (Sigma, cat. no. B-4501) 0.1 mg/ ml,L-carnitine (Sigma, cat. no. C-0283) 2 mg/ ml, ethanolamine (Sigma, cat.no. E-9508) 1 mg/ml , D-galactose (Sigma, cat. no. G-0625) 15 mg/ ml/,L-proline (Sigma, cat. no. P-0380) 7.76 mg/ ml , putrescine (Sigma, cat. no.P-7505) 16.1 mg/ ml , Na-pyruvate (Sigma, cat. no. P-5280) 25 mg/ ml ,Na-selenite (Sigma, cat. no. S-1382) 0.016 mg /ml , vitamin B12 (Sigma,cat. no. V-2876) 0.34 mg/ ml , zinc sulfate (Sigma, cat. no. Z-4750) 0.194mg /ml , catalase (Sigma, cat. no. C-40) 16 mg /ml , glutathione (Sigma,cat. no. G-6013) 1 mg/ ml, superoxide dismutase (Sigma, cat. no. S-2515)2.5 mg/ml ; ethanolic solutions: linoleic acid (Sigma, cat. no. L-1376)100 mg /ml , linolenic acid (Sigma, cat. no. L-2376) 100 mg /ml ,progesterone (Sigma, cat. no. P-8783) 0.63 mg/ ml , all-trans retinol(Sigma, cat. no. R-7632) 10 mg/ ml , retinylacetate (Sigma, cat. no. R-7882)10 mg/ ml , tocopherol (Sigma, cat. no. T-3251) 100 mg/ ml , tocopherolacetate (Sigma, cat. no. T-3001) 100 mg /ml. B27作用 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-s 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 hippo campal 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. 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.