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SF27 ( 含维生素A, 50×)
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为什么选择此级别
BioReagent,无菌,用于细胞培养 级 ，适用于对基线干扰要求严格的色谱和分析工作流程。
储存与运输
-20°C储存,避免反复冻融。超低温运输 。请查阅批次 COA 获取详细规格。
质量文档
SDS、COA、产品数据表及规格说明书均可下载。可通过批号查询获取批次 COA。
文献证明
在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。
概述
SF27 (含维生素A, 50×)，是一种优化的无血清添加剂，广泛用于支持胚胎，出生后和成体的海马神经元和其他中枢神经系统（CNS）神经元的生长、分化和活性维持。无论是悬浮的神经球还是贴壁单层培养，都是用于培养神经祖细胞和神经干细胞的理想选择，而不会诱导分化。SF27添加剂以50X液体形式提供，在神经元基础培养基中使用B-27添加剂，用于培养产前和胚胎神经元，以获得优化的活性和长期的存活率。也可用于培养神经来源的肿瘤细胞系，可有效地保证其活性。现也已广泛应用于各种乳腺癌、小肠、胃、肝脏等类器官的培养中。
储存条件
-20℃，有效期2年。
应用
用于大鼠海马神经元及多种中枢神经元细胞的生长和活性维持; 干细胞增殖及分化; 维持神经细胞来源的肿瘤细胞系的活性; 优化胚胎和出生后神经元的活性和长期存活率; 胚胎干细胞分化为神经元谱系(神经元和星形胶质细胞); 肿瘤类器官的培养。
使用方法
SF27 (含维生素A, 50×)是50X 浓度。使用前请用基础培养基稀释到1X。如准备50mL培养基： 将1mL的 B-27 无血清添加剂(50X)加入到49mL 的基础培养基中。
培养基的配置
由于L-谷氨酰胺在水溶液中不稳定，其分解产物对细胞具有毒性，神经元基础培养基和SF27中都未包含L-谷氨酰胺，因此在配制培养基时须另外添加L-谷氨酰胺。
1、置于4℃解冻本产品。
2、在使用前无菌添加2％本产品和0.5ML-谷氨酰胺至神经元基础培养基，配制成神经元完全培养基（注：下文中出现的“神经元完全培养基”即指此种添加SF27添加剂和L-谷氨酰胺的神经元基础培养基）。
3、注意：剩余的本产品可以等分成工作体积，并储存在-20℃。在以后的试验中根据需要解冻相应体积的本产品。避免 反复冻融本产品。
4、对于原代海马神经元培养，神经元完全培养基（由前述步骤制备）需要额外补充25μM的L-谷氨酸，在培养的第4 天以后的换液中应不再添加谷氨酸。配置好的完全培养基，可于2-8℃的避光保存一周。
细胞培养步骤
下列步骤已在大鼠胎儿原代皮层神经元中测试。
1、用无菌的冷的 0.05mg/L多聚赖氨酸水溶液包被培养表面（玻璃或细胞培养级塑料），每平方厘米表面使用0.15 mL，37 ℃放置过夜(16h)。
2、去除多聚赖氨酸溶液，并用无菌蒸馏水冲洗两次（须彻底清洗，因为多聚赖氨酸对细胞有毒性）。在超净工作台中 打开培养板的盖子通风，直到每个孔都完全干燥。培养板干燥后可以立即使用或在 4℃最多保存 2 周。
3、根据实验室标准操作流程从胚胎16-18days的大鼠中分离获得原代海马/皮层神经元细胞，或参考细胞株提供的说明书解冻冻存的原代大鼠神经元细胞。
4、将神经元完全培养基在37℃的水浴锅中预热，建议细胞接种密度为 2×10⁵ 个/孔（24 孔板为例）或根据自身实 验对密度进行调整。
5、在将接种了细胞的培养皿放置于 37ºC，5% CO₂ 的培养箱中培养。
6、在接种16-24h后，更换一半体积的新鲜的完全培养基，之后2-3天换液。
分离原代大鼠胚胎神经元
下列程序推荐用于培养受精18天的大鼠胚胎海马神经元和大脑皮层神经元。
1、从受精18天的大鼠胚胎中分离大脑皮层和双侧海马。
2、在预置了完全培养基的锥形管中收集所有的组织。放置，直到所有的组织都已解体。
3、使组织沉积在管的底部，然后小心地去除上清液，只留下刚好可覆盖组织的最少量的培养基。
4、在不含钙离子的培养基中，使用2mg/L过滤灭菌的木瓜蛋白酶溶液，在 37°C 下酶解组织约 30min，期间轻摇 锥形管以帮助降解，5min/次。每对海马组织使用 2mL 酶溶液。
5、加入两倍体积的完全培养基以恢复二价阳离子的浓度，停止酶解。
6、使未解离的组织沉降至管底（约 2min），然后把上清液转移到 15mL 离心管中，离心，150×g，5min。
7、在 1mL神经元完全培养基中重悬沉淀物，取 10μL 进行细胞计数。后续的操作按照“复苏和培养冻存的神经元” 的第 8-10 步骤进行。
复苏和培养冻存的神经元
重要提示：原代神经元细胞将会贴附在裸露的塑料或玻璃表面，建议事先用神经元完全培养基冲洗所有的塑料和玻璃器皿的内表面，以获得最高的回收率和产量。由于细胞从冻存状态复苏时极其脆弱，请在整个操作过程中不要震荡或离心细胞。 我们建议每次只解冻一管细胞。务必减少从液氮中转移冻存管到 37℃水浴中的操作时间。
1、在解冻细胞之前，用神经元完全培养基冲洗无菌的15mL锥形管，然后在超净工作台中通风晾干。
2、从液氮中取出冻存管时可稍微拧松管帽以释放压力，然后再拧紧。
3、在 37℃水浴中轻柔搅拌冻存管以快速解冻细胞（小于2min）。当观察到冻存管中只有很少量的冰晶存在时（触摸冻存管仍然感觉是冷的）从水浴中取出。
4、在工作台台面上轻敲冻存管以使管内液体沉降到管底。使用事先用神经元完全培养基冲洗并干燥过的1mL吸头极 其轻柔地将细胞转移到事先冲洗并干燥的 15mL 锥形管中。
5、用1mL 预热的神经元完全培养基冲洗冲洗冻存管，以每秒一滴的速度极其缓慢地加入到 15mL锥形管中的细胞 里。每加一滴都轻柔地转动锥形管一次。不要一次即把全部培养基加到锥形管中。
6、慢慢地逐滴加入另外 2mL 预热的完全培养基，使管内的液体体积为 4mL。用 1mL 吸头轻柔地混合液体，注意 不要造成任何气泡。
7、用一个事先冲洗干燥过的吸头吸取 10μL细胞悬液加入到含有 10μL，0.4%台盼蓝的微量离心管中，轻敲管壁 以混匀溶液。使用手动的细胞计数方法测定活细胞密度。解冻的细胞的活率应超过50%。
8、在已经事先用多聚赖氨酸包被的 24 孔板中每孔中接种大约 2×10⁵ 个细胞或按所需的 细胞密度接种。加入预热的神经元完全培养基将细胞悬液稀释到每孔 500μL。按照“细胞培养步骤”中的 5-6 步骤进行神 经元的培养。在 37℃，含5% CO₂ 的培养箱中培养。
注意事项
1、本产品在使用前在4℃过夜解冻。
2、使用样品时将产品充分混匀进行添加。
3、使用样品时应注意无菌操作，避免污染。
4、为保持产品的最佳使用效果，不宜将其过夜放置于室温或较高的温度环境中。 5、请于保质期内使用本产品，超出保质期，请放弃使用。
SF27 (with Vitamin A, 50×) is an optimized serum-free supplement. It is widely used to support the growth, differentiation and activity maintenance of hippocampal neurons and other central nervous system (CNS) neurons derived from embryos, postnatal and adult tissues. It is an ideal choice for culturing neural progenitor cells and neural stem cells in both suspended neurospheres and adherent monolayer cultures without inducing differentiation.Supplied as a 50× liquid concentrate, SF27 is used together with B-27 supplement in neuronal basal medium for the culture of prenatal and embryonic neurons to achieve optimized cell activity and long-term survival rate. It can also be applied to neuron-derived tumor cell lines to effectively sustain cell viability. Currently, it has been extensively used for the culture of various organoids including breast, small intestine, stomach and liver organoids.
Storage
Store at -20 °C for 2 years.
Applications
Maintain the growth and viability of rat hippocampal neurons and various central neurons
; Support the proliferation and differentiation of stem cells; Sustain the activity of neuron-derived tumor cell lines; Optimize the activity and long-term survival rate of embryonic and postnatal neurons; Facilitate the differentiation of embryonic stem cells into neuronal lineages (neurons and astrocytes); Culture tumor organoids
Usage Instructions
SF27 (with Vitamin A, 50×) is a 50× concentrated solution. Dilute it to 1× with basal medium prior to use.
Example for preparing 50 mL medium: Add 1 mL of B-27 serum-free supplement (50×) into 49 mL of basal medium.
Medium Preparation
L-Glutamine is unstable in aqueous solution, and its degradation products are toxic to cells. Neither neuronal basal medium nor SF27 contains L-glutamine, so additional L-glutamine must be supplemented during medium preparation.
1. Thaw the product at 4 °C.
2. Aseptically add 2% of this product and 0.5 mM L-glutamine into neuronal basal medium to prepare complete neuronal medium.
3. Note: The term "complete neuronal medium" hereinafter refers to neuronal basal medium supplemented with SF27 and L-glutamine.
Note: Aliquot the remaining product into working volumes and store at -20 °C. Thaw the required volume for subsequent experiments. Avoid repeated freeze-thaw cycles.
4. For primary hippocampal neuron culture, supplement the aforementioned complete neuronal medium with additional 25 μM L-glutamic acid. Do not add glutamic acid during medium replacement starting from the 4th day of culture. The prepared complete medium can be stored away from light at 2–8 °C for up to one week.
Cell Culture Protocol
The following procedures have been validated using primary cortical neurons isolated from rat embryos.
1. Coat culture surfaces (glass or cell-culture grade plastic) with sterile cold 0.05 mg/L poly-L-lysine aqueous solution at 0.15 mL per square centimeter. Incubate overnight (16 hours) at 37 °C.
2. Remove the poly-L-lysine solution and rinse the surfaces twice with sterile distilled water. Rinse thoroughly as poly-L-lysine is cytotoxic. Leave the culture plates uncovered in a biosafety cabinet until all wells are completely dry. The plates can be used immediately or stored at 4 °C for a maximum of 2 weeks.
3. Isolate primary hippocampal/cortical neurons from rat embryos at embryonic day 16–18 following standard laboratory protocols, or thaw cryopreserved primary rat neurons in accordance with the instructions for the cell line.
4. Pre-warm the complete neuronal medium in a 37 °C water bath. The recommended seeding density is 2 × 10⁵ cells per well (for 24-well plates), which can be adjusted according to experimental requirements.
5. Place the seeded culture vessels in an incubator at 37 °C with 5% CO₂ for cultivation.
6.Replace half of the medium with fresh complete medium 16–24 hours after seeding, and perform medium replacement every 2 to 3 days thereafter.
Isolation of Primary Rat Embryonic Neurons
This protocol is recommended for culturing hippocampal and cortical neurons from rat embryos at embryonic day 18.
1. Dissect cerebral cortex and bilateral hippocampi from rat embryos at embryonic day 18.
2. Collect all tissues into a conical tube pre-filled with complete medium and let the tissues settle.
3. Allow the tissues to sediment at the bottom of the tube. Carefully aspirate the supernatant, leaving only a minimal volume of medium to cover the tissues.
4. Digest the tissues with 2 mg/L filter-sterilized papain solution in calcium-free medium at 37 °C for approximately 30 minutes. Gently invert the conical tube every 5 minutes to facilitate digestion. Use 2 mL of papain solution per pair of hippocampi.
5. Add complete medium at twice the volume of the enzyme solution to restore divalent cation levels and terminate digestion.
6. Let undigested tissues settle for about 2 minutes, then transfer the supernatant into a 15 mL centrifuge tube and centrifuge at 150 × g for 5 minutes.
7. Resuspend the cell pellet in 1 mL of complete neuronal medium. Pipette 10 μL of cell suspension for cell counting. Follow steps 8 to 10 in Thawing and Culturing Cryopreserved Neurons for subsequent operations.
Thawing and Culturing Cryopreserved Neurons
Important Notes: Primary neurons tend to adhere to bare plastic or glass surfaces. It is recommended to rinse the inner surface of all plastic and glassware with complete neuronal medium in advance to maximize cell recovery and yield. Cryopreserved neurons are extremely fragile upon thawing. Do not shake or centrifuge the cells throughout the entire process. Thaw only one cryovial at a time, and minimize the time taken to transfer cryovials from liquid nitrogen to a 37 °C water bath.
1. Rinse a sterile 15 mL conical tube with complete neuronal medium before thawing cells, then air-dry it in a biosafety cabinet.
2. Loosen the cap slightly to release pressure when taking the cryovial out of liquid nitrogen, then tighten the cap again.
3. Immerse the cryovial in a 37 °C water bath and swirl gently to thaw cells rapidly (within 2 minutes). Remove the vial from the water bath when only a small amount of ice crystals remain (the vial still feels cold to the touch).
4. Tap the cryovial gently on the bench to collect all liquid at the bottom. Use a 1 mL pipette tip (pre-rinsed and dried with complete neuronal medium) to transfer the cells extremely gently into the pre-treated 15 mL conical tube.
5. Rinse the cryovial with 1 mL pre-warmed complete neuronal medium, and add the medium dropwise (one drop per second) to the cells in the 15 mL tube. Gently swirl the tube after each drop. Do not add all medium at once.
6. Slowly add another 2 mL pre-warmed complete medium dropwise to bring the total volume to 4 mL. Mix gently with a 1 mL pipette tip and avoid bubble formation.
7. Pipette 10 μL of cell suspension into a microcentrifuge tube containing 10 μL of 0.4% trypan blue. Tap the tube gently to mix, then count viable cells manually. The viability of thawed cells should be above 50%.
8. Seed approximately 2 × 10⁵ cells per well into pre-coated 24-well plates , or adjust the seeding density as required. Dilute the cell suspension with pre-warmed complete neuronal medium to a final volume of 500 μL per well. Culture the neurons following steps 5 and 6 in Cell Culture Protocol in an incubator at 37 °C with 5% CO₂.
Precautions
1. Thaw the product overnight at 4 °C before use.
2. Mix the product thoroughly prior to use.
3. Perform all operations under aseptic conditions to prevent contamination.
4. Do not leave the product at room temperature or elevated temperature overnight to ensure optimal performance.
5. Use the product within the stated shelf life. Discard expired products.
规格
别名
培养基补充剂SF27
英文别名
B27(50×) | SF27 (50×) Medium Supplement | SF27 50× Culture Supplement | SF27 Supplement, 50× Concentrate
规格或纯度
BioReagent, 用于细胞培养, 无菌, 50×， 含维生素A
稳定性与储存
Store at -20°C long term (24months). Avoid freeze/thaw cycle.
英文名称
SF27 (with Vitamin A, 50×)
储存条件
-20°C储存,避免反复冻融
运输条件
超低温运输
产品线
生物产品
名称和识别符
分子类型
生物试剂/缓冲液
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C of A & Other Certificates(BSE/TSE, COO):
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0636932 |
分析证书 | S1523188 |
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