B-27添加剂 (无胰岛素, 50×)是初始B-27添加剂 (含胰岛素)的定制版本。B-27无胰岛素添加剂是一种优化的无血清添加剂，设计用于支持在存在胰岛素的情况下会引起干扰的应用中海马和其他CNS神经元的生长。B-27无胰岛素添加剂以50×液体形式提供，适合与Neurobasal培养基或Neurobasal -A培养基配合使用。
应用
用于大多数神经元细胞应用。
使用方法
B-27添加剂 (无胰岛素, 50×)是50×浓度。使用前请用基础培养基 (如Neurobasal)稀释到1×。如准备50 mL培养基： 将1 mL的B-27添加剂 (无胰岛素, 50×)加入到49 mL的基础培养基中。
1. 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B-27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0.5 ml L-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B-27无胰岛素添加剂和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25 μM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。
2. 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0.05 mg/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16-18 days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°C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 days换液。
3. 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2 mg/L过滤灭菌的木瓜蛋白酶溶液，在37°C下酶解组织约30 min，期间轻摇锥形管以帮助降解，5 min/次。每对海马组织使用2 mL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 2 min)，然后把上清液转移到15 mL离心管中，离心，150× g，5 min。
(7) 在1 mL神经元完全培养基中重悬沉淀物，取10 μL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。
4. 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°C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15 mL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°C水浴中轻柔搅拌冻存管以快速解冻细胞 (小于 2 min)。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1 mL吸头极其轻柔地将细胞转移到事先冲洗并干燥的15 mL锥形管中。
(5) 用1 mL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15 mL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2 mL预热的完全培养基，使管内的液体体积为4 mL。用1 mL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10 μL细胞悬液加入到含有10 μL，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 (参见“细胞培养步骤”)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500 μL。按照“细胞培养步骤”中的5-6步骤进行神经元的培养。在37°C，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. 使用产品时将产品充分混匀进行添加。
3. 如果不立即使用，请按需分装并储存在-20 ℃下，请不要反复冻融。
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. 使用产品时应注意无菌操作，避免污染。
6. 请于保质期内使用本产品，超出保质期，请放弃使用。
B-27 Supplement (minus insulin, 50×) is a customized version of the original B-27 Supplement (with insulin). As an optimized serum-free additive, B-27 Supplement minus insulin is designed to support the growth of hippocampal and other CNS neurons in applications where the presence of insulin may cause interference. Supplied as a 50× liquid formulation, it is compatible with Neurobasal Medium or Neurobasal-A Medium.
Applications
Suitable for most neuronal cell culture applications.
Instructions for Use
B-27 Supplement (minus insulin, 50×) is a 50× concentrated solution. Dilute to 1× with basal medium such as Neurobasal before use.Preparation example for 50 mL medium: Add 1 mL of B-27 Supplement (minus insulin, 50×) into 49 mL of basal medium.
1. Medium Preparation
L-Glutamine is unstable in aqueous solution, and its degradation products are cytotoxic to cells. Neither neuronal basal medium nor B-27 contains L-glutamine; therefore, additional L-glutamine must be supplemented during medium preparation.
(1) Thaw the product at 4 ℃.
(2) Aseptically add 2% of this product and 0.5 mL L-glutamine into neuronal basal medium to prepare complete neuronal medium. (Note: The term "complete neuronal medium" hereinafter refers to neuronal basal medium supplemented with B-27 Supplement minus insulin and L-glutamine.)
(3) Note: Aliquot the remaining product into working volumes and store at -20 ℃. Thaw the required volume for subsequent experiments as needed. Avoid repeated freeze-thaw cycles.
(4) For primary hippocampal neuron culture, supplement the prepared complete neuronal medium with an additional 25 μM L-glutamate. Do not add glutamate during medium replacement from day 4 of culture onwards. The prepared complete medium can be stored at 2–8 ℃ in the dark for up to one week.
2. Cell Culture Protocol
The following protocol has been validated in primary cortical neurons from embryonic rat fetus.
(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 cm² surface area, and incubate overnight (16 h) at 37 ℃.
(2) Remove the poly-L-lysine solution and rinse twice with sterile distilled water (thorough rinsing is required, as poly-L-lysine is cytotoxic). Place the culture plate uncovered in a biosafety cabinet for air drying until all wells are completely dry. The dried plate can be used immediately or stored at 4 ℃ for up to 2 weeks.
(3) Isolate primary hippocampal/cortical neurons from rat embryos at embryonic day 16–18 following laboratory standard operating procedures, or thaw cryopreserved primary rat neurons according to the manufacturer’s instructions.
(4) Pre-warm complete neuronal medium in a 37 ℃ water bath. The recommended seeding density is 2×10⁵ cells per well (for 24-well plates), which may be adjusted according to experimental requirements.(5) Place the seeded culture dishes in a humidified incubator at 37 ℃ with 5% CO₂ for cultivation.
(6) Replace half of the medium with fresh complete neuronal medium 16–24 h after seeding, followed by medium replacement every 2–3 days thereafter.
3. Isolation of Primary Rat Embryonic Neurons
This protocol is recommended for the culture of hippocampal and cerebral cortical neurons from rat embryos at embryonic day 18.
(1) Dissect cerebral cortex and bilateral hippocampus from rat embryos at embryonic day 18.
(2) Collect all tissues in a conical tube pre-filled with complete medium and allow tissue dissociation to proceed naturally.
(3) Allow tissues to settle to the bottom of the tube, then carefully remove the supernatant, leaving only a minimal volume of medium to just cover the tissues.
(4) Digest tissues in calcium-free medium with 2 mg/L filter-sterilized papain solution at 37 ℃ for approximately 30 min. Gently rock the conical tube every 5 min to facilitate dissociation. Use 2 mL of papain solution per pair of hippocampal tissues.
(5) Add twice the volume of complete medium to restore divalent cation concentration and terminate enzymatic digestion.
(6) Allow undissociated tissue to settle for about 2 min. Transfer the supernatant into a 15 mL centrifuge tube and centrifuge at 150× g for 5 min.
(7) Resuspend the cell pellet in 1 mL of complete neuronal medium, take 10 μL for cell counting, and follow steps 8–10 in Recovery and Culture of Cryopreserved Neurons for subsequent operations.
4. Recovery and Culture of Cryopreserved Neurons
Important Note: Primary neurons adhere to bare plastic or glass surfaces. It is recommended to pre-rinse the inner surface of all plastic and glassware with complete neuronal medium to maximize cell recovery and viability. Neurons are extremely fragile upon thawing; do not shake or centrifuge cells throughout the entire procedure. Thaw only one vial at a time, and minimize the time interval between transferring cryovials from liquid nitrogen to a 37 ℃ water bath.
(1) Before thawing cells, rinse a sterile 15 mL conical tube with complete neuronal medium and air-dry it in a biosafety cabinet.
(2) Loosen the cryovial cap slightly immediately after taking it out of liquid nitrogen to release pressure, then tighten the cap again.
(3) Thaw the cryovial rapidly in a 37 ℃ water bath with gentle agitation (within 2 min). Remove the vial when only a small amount of ice crystals remain (the vial still feels cold to the touch).
(4) Tap the cryovial gently on the cabinet surface to settle the liquid to the bottom. Use a 1 mL pipette tip pre-rinsed and dried with complete neuronal medium to transfer the cell suspension extremely gently into the pre-rinsed 15 mL conical tube.
(5) Rinse the cryovial with 1 mL pre-warmed complete neuronal medium, and add it dropwise at a rate of one drop per second to the cell suspension in the 15 mL tube. Gently rotate 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. Gently mix with a 1 mL pipette tip without generating air bubbles.
(7) Pipette 10 μL cell suspension and mix with 10 μL 0.4% trypan blue in a microcentrifuge tube by gently tapping the tube. Determine viable cell density via manual cell counting. The cell viability after thawing should exceed 50%.
(8) Seed approximately 2×10⁵ cells per well into 24-well plates pre-coated with poly-L-lysine (refer to Cell Culture Protocol), and dilute the cell suspension to a final volume of 500 μL per well with pre-warmed complete neuronal medium. Culture neurons following steps 5–6 in Cell Culture Protocol at 37 ℃ with 5% CO₂.
Precautions
1. Thaw the product overnight at 4 ℃ before use.
2. Mix the product thoroughly before addition.
3. For immediate unused portion, aliquot appropriately and store at -20 ℃. Avoid repeated freeze-thaw cycles.
4. For optimal performance, do not leave the product at room temperature or elevated temperature overnight.
5. Strictly perform aseptic operation during use to avoid contamination.
6. Use the product within the shelf life; discard any expired product.
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