计算溶液所需的质量、体积或浓度。
外泌体提取纯化试剂盒 (组织)
有货
为什么选择此级别
BioReagent,无菌 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
室温,-20°C储存。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
是否无菌：是
外泌体是由细胞分泌的包含 RNA 和蛋白质的小囊泡 (30-150 nm)，在血液、唾液、尿液及乳汁等体液中大量存在。外泌体被认为具有细胞间信使的功能，在特定细胞之间传递它们的效应物或信号分子；然而其构造、效应物组成 以及所参与的生物学通路目前尚不明晰。外泌体的生物学功能研究中需要分离完整的外泌体颗粒，而传统超速离心方法步骤繁琐、硬件要求高、操作难度大。该外泌体提取纯化试剂盒，组分经过优化处理，适用于多种组织的外泌体提取 (包括脑、心、 肝、肺、肌肉、脾、淋巴结、胸腺、胚胎、肿瘤等组织)， 可快速高效地获得高纯度外泌体颗粒，可用于电镜分析、 NTA 粒径分析、核酸分析、蛋白分析、细胞学实验和动物实验等。
产品组成
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自备材料：
高速离心机，涡旋振荡器，2 mL 离心管，1.5 mL 离心管，1×PBS 缓冲液（无菌）。
操作规程：
一、 样品预处理
1、 提前将溶液 A2 置于 4℃或冰上融化并混匀；
2、 组织剪碎：在消毒器皿（置于冰上）中将组织剪切成 1 mm³～3 mm³ 左右的碎组织块 （剪切得越小越好）；
3、 组织碎片清洗：将组织碎块转移至离心管中，加入不少于 10 倍体积的 1×PBS 缓冲液，振荡混匀后 300 ×g（~2,000 rpm*）离心 10 min，弃上清；
*为约 7 cm 有效离心半径的小离心机换算（≤2 mL 离心管），下同。
4、 组织消化：按照每 0.1 g 组织块加入 1 mL Solution A2 的比例，将组织块与溶液 A2 混匀在 2 mL 离心管中，横放于 37℃、80 rpm 恒温摇床上孵育 20 min（或在 37℃水浴锅中孵育 30 min，每 5 min 混匀一次）；
5、 离心取上清：反应液以 8,000 ×g（~10,100 rpm）离心 10 min，将上清液转移至新的离心管中；
6、 离心去杂质碎片：将离心上清液转移至新的离心管中，于 4℃以 12,000 ×g（~12,400 rpm）离心 10 min，去除样品中的杂质碎片；
7、 上清液转移：去除杂质碎片的上清液转移到新离心管中。
二、 提取外泌体
1、 加溶液 B2 试剂：上一步获得的上清液中加入 1/4体积的溶液 B2 试剂；
2、 溶液混合：将离心管盖紧，通过涡旋振荡器混匀 1 min，再放置于 4℃静置 1 h 以上（注：增加静置时间可提高外泌体得率，但不可超过 24 h）；
3、 沉淀外泌体：取出装有混合液的离心管于 4℃以 12,000 ×g（~12,400 rpm）离心 30 min，弃上清，沉淀中富含外泌体颗粒（尽可能吸尽上清液）；
4、 再次离心：将含有沉淀的离心管再次于 4℃以 12,000 ×g（~12,400 rpm）离心 2 min，弃上清（注：尽可能吸尽上清液）；
5、 外泌体重悬：取合适量的 1×PBS 均匀吹打离心沉淀物，待其溶解后，将重悬液转移至新的 1.5 mL 离心管中（建议每 0.1 g 组织用 200 μL 左右 1×PBS 重悬）；
6、 收获外泌体颗粒：将含有重悬液的1.5 mL 离心管于4℃以 12,000 ×g（~12,400 rpm）离心 2 min，保留上清液，其中富含外泌体颗粒（注：若沉淀较多，可 12,000×g，2 min 离心多次至无明显沉淀，每次取上清液）。
三、 纯化外泌体
1、 纯化外泌体：将收获的外泌体颗粒粗品转入外泌体纯化滤器（EPF 柱）上室中，于 4℃以 3,000 ×g（~6,200 rpm）离心 10 min，离心后收集 EPF 柱管底的液体，此液体即为纯化后的外泌体颗粒（注：EPF柱不可重复使用）；
2、 外泌体的保存：纯化后的外泌体以合适体积进行分装冻存于-80℃低温冰箱中，以备后续实验使用。
注意：本产品仅用于生命科学研究，不得用于医学诊断及其他用途。
常见问题及解决方法：
一、 产品试剂保存问题：
1、 溶液 A2 使用前后应保存于-20℃条件下，建议分装后使用，应避免反复冻融；
2、溶液 B2 始终保存在室温条件下即可，温度过低可能使该溶液中出现白色结晶，且难以复溶（需要高温）， 少量沉淀不影响提取效果。
二、 纯化柱使用问题：
1、 一般情况下，每个样品按照说明书操作，“3,000 ×g （~6,200 rpm）离心 10 min”即可一次性纯化完毕。 如果上柱中仍有液体残留，将纯化柱转换方向放置于离心机中（区别于上一次放置方向即可），再次重复离心；
2、 若通过上述操作，上柱中仍有较多液体残留，请将上柱中的液体转移至新的纯化柱中，继续重复离心操作，直至上柱液体全部转移至下柱中。
三、 外泌体得率问题：
一般情况下，0.1 g 组织样品（以心肌组织为例）可以提取出 100~200 μg 外泌体，因不同组织外泌体含量不同，得率会有一定差异。
Exosomes are small‑sized vesicles (30‑150 nm) secreted by cells, containing RNA and proteins, and are abundantly present in body fluids such as blood, saliva, urine and milk. Exosomes are regarded as intercellular messengers that deliver their effectors or signaling molecules between specific cells. Nevertheless, their structure, effector composition and involved biological pathways remain poorly understood. Research on the biological functions of exosomes requires isolation of intact exosome particles. Conventional ultracentrifugation is plagued by complicated workflows, high hardware requirements and demanding operation. This exosome isolation and purification kit features optimized components for exosome extraction from a wide range of tissues (including brain, heart, liver, lung, muscle, spleen, lymph node, thymus, embryo, tumor and other tissues). It enables rapid and efficient acquisition of high‑purity exosome particles, which are compatible with electron microscopy, NTA particle‑size analysis, nucleic acid analysis, protein analysis, cytological assays and animal experiments.
Components
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Customer-Supplied Materials
High-speed centrifuge, vortex mixer, 2 mL centrifuge tubes, 1.5 mL microcentrifuge tubes, sterile 1×PBS buffer.
Standard Operating Procedure
I. Sample Pretreatment
1.Thaw Solution A2 at 4 °C or on ice in advance and mix thoroughly.
2.Tissue mincing: Place sterilized labware on ice and cut tissues into small pieces of approximately 1 mm³ ~ 3 mm³ (smaller fragments yield better results).
3.Wash tissue fragments: Transfer tissue pieces to a centrifuge tube, add sterile 1×PBS buffer at no less than 10-fold tissue volume, vortex to mix, then centrifuge at 300 × g (~2,000 rpm*) for 10 min; discard supernatant.
*RPM conversion for small centrifuges with an effective radius of ~7 cm (for tubes ≤2 mL), applicable throughout the protocol.
4.Tissue digestion: Mix tissue fragments with Solution A2 in a 2 mL centrifuge tube at a ratio of 1 mL Solution A2 per 0.1 g tissue. Lay the tube horizontally on a thermostatted shaker at 37 °C with shaking speed of 80 rpm and incubate for 20 min. Alternatively, incubate in a 37 °C water bath for 30 min and invert to mix once every 5 min.
5.Centrifuge and collect supernatant: Centrifuge the digested mixture at 8,000 × g (~10,100 rpm) for 10 min, then transfer supernatant to a new centrifuge tube.
6.Remove miscellaneous debris by centrifugation: Transfer the supernatant to a fresh tube, centrifuge at 12,000 × g (~12,400 rpm) for 10 min at 4 °C to eliminate fine impurities.
7.Transfer clarified supernatant: Move the debris-free supernatant to a new centrifuge tube.
II. Exosome Extraction
1.Add Solution B2 reagent: Supplement the clarified supernatant with Solution B2 at a volume ratio of 4:1 (1/4 volume of supernatant).
2.Mix and incubate: Tightly cap the tube, vortex for 1 min to homogenize thoroughly, then incubate at 4 °C for a minimum of 1 h.
Note: Extended incubation can improve exosome yield, but the incubation time shall not exceed 24 h.
3.Pellet exosomes: Centrifuge the mixture tube at 12,000 × g (~12,400 rpm) for 30 min at 4 °C. Discard supernatant; the pellet contains concentrated exosome particles. Aspirate supernatant completely as far as possible.
4.Secondary centrifugation: Re-centrifuge the tube containing the pellet at 12,000 × g (~12,400 rpm) for 2 min at 4 °C, then thoroughly aspirate and discard residual supernatant.
Note: Remove supernatant to the maximum extent.
5.Resuspend exosomes: Add an appropriate volume of sterile 1×PBS to the pellet and gently pipette up and down until the pellet is fully dissolved. Transfer the resuspension to a new 1.5 mL microcentrifuge tube. Recommended resuspension volume: ~200 μL sterile 1×PBS per initial 0.1 g tissue.
6.Harvest crude exosome particles: Centrifuge the 1.5 mL tube with exosome resuspension at 12,000 × g (~12,400 rpm) for 2 min at 4 °C. Retain the supernatant which contains crude exosomes.
Note: If visible precipitate remains, repeat centrifugation at 12,000 × g for 2 min until no obvious pellet is observed; retain supernatant after each spin.
III. Exosome Purification
1.Purify exosomes: Load crude exosome suspension into the upper chamber of Exosome Purification Filter (EPF column). Centrifuge at 3,000 × g (~6,200 rpm) for 10 min at 4 °C. Collect the eluate at the bottom collection tube of EPF column, which is purified exosome solution.
Note: EPF columns are disposable and cannot be reused.
2.Exosome storage: Aliquot purified exosomes into appropriate volumes and store at -80 °C freezer for downstream experiments.
Precautions
This product is intended solely for life science research purposes. It must not be used for medical diagnosis or clinical applications.
Frequently Asked Questions & Troubleshooting
I. Storage of Kit Reagents
1.Store Solution A2 at -20°C before and after use. Aliquot into small portions for repeated use to avoid repeated freeze-thaw cycles.
2.Solution B2 can be stored at room temperature permanently. Low temperature may induce white crystalline precipitation in Solution B2 which is hard to redissolve (requires high temperature treatment). Minor precipitation does not interfere with exosome extraction efficiency.
II. Operation of Purification Column
1.Under standard conditions, one single centrifugation at 3,000 × g (~6,200 rpm) for 10 min can complete full purification for each sample. If liquid residue remains in the upper column, flip the column to reverse its orientation in the centrifuge and repeat centrifugation.
2.If abundant liquid still remains after the above operation, transfer residual liquid from the upper column to a brand-new EPF column and repeat centrifugation until all liquid flows down to the collection tube.
III. Exosome Yield
In general, 0.1 g tissue sample (e.g., cardiac muscle tissue) yields 100–200 μg exosomes. Exosome yield varies among different tissue types due to intrinsic differences in exosome secretion levels.
规格
规格或纯度
BioReagent, 无菌, 无核酸酶
英文名称
Exosome Isolation and Purification Kit (Tissue)
应用
Exosome Isolation and Purification
样品类型
组织
储存与运输
储存条件
室温,-20°C储存
运输条件
超低温运输
稳定性与储存
各组分在相应的储存条件下保质期24个月。
组分与储存
| 储运单元货号 | 储运单元名称 | 组分货号 | 组分名称 | 物理外观 | 2T | 储存温度 |
| E1528051NT | 外泌体提取纯化试剂盒（组织）(常温组分) | E1528051A | 溶液 B2 | 液体 | 1.2 mL | 室温 |
| E1528051B | 外泌体纯化滤器 | - | 2 EA | 室温 | ||
| E1528051DT | 外泌体提取纯化试剂盒（组织）(-20℃组分) | E1528051C | 溶液 A2 | 液体 | 5 mL | -20℃. |
注：发货时各组分依据储存温度（室温/-20℃）独立包装，差异化温控运输以保障产品性能。同一订单含多温区物料可能分批次送达，请以实际收货为准。收货后按组分表标注温度分类存放，有效期自生产之日起 24 个月。
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溶液计算器
产品仅提供给经过验证的企业、机构和合格的专业人员用于研发。不适用于人类、动物、诊断或治疗。
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