BioReagent 级 ，适用于对基线干扰要求严格的色谱和分析工作流程。
室温,-80℃储存,避免反复冻融。常规运输,特低温运输 。请查阅批次 COA 获取详细规格。
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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。
植物囊泡在细胞间通讯、免疫调节及跨物种调控中具有重要研究价值，但其提取面临植物组织成分复杂（如多糖、酚类干扰物）和细胞壁致密的技术瓶颈，导致得率低、纯度不足。本试剂盒专为多汁植物设计，可高效释放植物组织中的囊泡，并去除植物组织中的非囊泡成分，获得的植物囊泡可用于电镜分析、NTA 粒径分析、核酸分析、蛋白分析、细胞学实验和动物实验等。
组分与储存
① 发货时各组分依据储存温度（RT/-80℃）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 12 个月。 ② *为无菌，**为无菌无酶 |
自备材料：
一、实验材料/耗材
1、 削皮刀、水果刀、玻璃砧板等（可以把待提取植物处理成方便榨汁的小块的用具）；
2、 400 mL 烧杯 1~2 个；
3、 50 mL 离心管、1.5 mL EP 管若干；
4、 pH 试纸，1~10（广泛），5.5~8.5（精密）。
二、实验设备
1、 电子秤；
2、 碾磨榨汁机（推荐使用，可搜索原汁机购买）；
3、 4℃高速离心机；
4、 水浴锅；
5、 涡旋振荡器。
三、实验试剂
1×PBS 溶液（无菌）
操作流程：
一、 榨汁前准备
1、 榨汁机消毒：将榨汁机各部件拆分开，喷洒 75%酒精仔细擦拭后，放入生物安全柜紫外消毒至少 20 min；
2、 样品准备：按照试剂盒处理量准备待提取样品；
注：2 T的试剂盒可处理植物汁液20 mL。
3、 样品处理：将新鲜的待提取植物洗净外表皮后，用无尘布擦干，放入生物安全柜紫外消毒至少 20 min 后，将植物切成方便榨汁的小块；
注：a. 皮厚的植物（如葛根）需要先洗净外皮、消毒后去皮切小块或削成薄片；
b. 皮薄的植物（如三七、地黄、姜黄）可以洗净外皮，消毒后直接切小块，准备榨汁。
二、 榨汁
1、 榨汁机准备：在生物安全柜下，将完成消毒的榨汁机部件按照榨汁机说明书组装；
2、 榨汁：将切好的植物小块投入榨汁机榨汁，质地偏硬的植物注意少量多次，收集植物汁液，榨汁完成。
注：如需控制内毒素，榨汁过程请在生物安全柜内进行。
三、 离心去杂
1、 低速离心去杂：将榨汁后的汁液于 4℃，5,000 ×g，10 min 离心，收集上清；
2、 高速离心去杂：将低速离心后收集的上清于 4℃，10,000 ×g，10 min 离心，收集上清。
四、 提取囊泡
1、 上清液预处理：在去除杂质后的植物汁液中加入混匀的溶液A ，20 mL离心去杂后的植物汁液加入200 μL溶液A，其他剂量可按添加比例换算。
注：溶液A使用前需混匀。
2、 混合孵育：加入溶液A后盖好管盖，颠倒混匀 8~10 次，将混合液 37℃水浴 16 h 左右（过夜）；
3、 调 pH：将孵育好的混合液取出，在生物安全柜下用试剂盒自带的 1M 氢氧化钠 和 1%柠檬酸将混合液 pH 调至 7.0；
4、 高速离心去杂：将调 pH 后的上清 4℃，10,000 ×g，20 min 离心后收集上清；
5、 过滤：将收集到的上清依次经 1 μm 滤膜和 0.45 μm滤膜过滤；
注：如需获得 30-1000 nm 植物囊泡，可只进行 1 μm滤膜过滤，不进行 0.45 μm 滤膜过滤。
6、加提取试剂：取上步离心上清液加入溶液B，20 mL离心去杂后的植物汁液加入5 mL溶液B，其他剂量请等比例换算。
7、 二次混合孵育：加入溶液B后拧紧管盖，通过涡旋振荡器混匀 1 min，将混合液 4℃静置 4 h 左右；
8、 沉淀植物囊泡：取孵育好的混合液，于 4℃，10,000×g，60 min 离心，弃上清，将含有沉淀的离心管再次于 4℃，10,000 ×g，2 min 离心后，吸尽上清；
注：沉淀中富含植物囊泡，需尽可能吸尽上清液。
9、 植物囊泡重悬：取合适量的 1×PBS 均匀吹打离心沉淀，待其溶解后，将重悬液转移至新的 1.5 mL EP 管中（建议每 20 mL 植物汁液用 400 μL 左右 1×PBS重悬）；
10、 植物囊泡收获：将含有重悬液的 1.5 mL EP 管于 4℃，12,000 ×g 离心 2 min，保留上清液，其中富含植物囊泡颗粒；
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。
五、 纯化和保存植物囊泡
1、 纯化植物囊泡：将收获的植物囊泡颗粒粗品转入外泌体纯化滤器（EPF 柱）上室中，于4℃，3,000 ×g 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植物囊泡颗粒；
注：a. EPF 柱不可重复使用；
b. 如需获得大颗粒植物囊泡，可不进行该步骤。
2、 植物囊泡保存：将纯化后的植物囊泡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。
注意事项
1、 2 T 试剂盒内的溶液 A ，收到后尽快放-80℃保存，使用前 4℃解冻，解冻后尽快用完，禁止反复冻融。
2、溶液 A 静置有棕黄色沉淀，属于正常现象，使用前需混匀。
3、 若对内毒素有严格要求，榨汁步骤请严格在生物安全柜下进行，关键用品和仪器注意消毒后使用。
4、 纯化后的植物囊泡可保存在-80℃冰箱中，避免反复冻融导致植物囊泡破裂。
Plant‑derived extracellular vesicles possess considerable research value in intercellular communication, immune regulation and cross‑species modulation. Nevertheless, their isolation is technically hindered by complex plant tissue components (e.g., polysaccharides, phenolic interferents) and dense cell walls, which result in low yield and insufficient purity. This kit is specifically designed for succulent‑fresh plant samples. It enables efficient release of vesicles from plant tissues and removal of non‑vesicular plant components. The resultant plant extracellular vesicles are compatible with transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), nucleic acid analysis, protein analysis, cytological assays and animal experiments.
Contents & Storage
1. Each component is individually packaged according to its storage temperature (RT/-80 °C) upon shipment, with differentiated temperature‑controlled transportation to guarantee product performance. Orders containing materials requiring multiple temperature zones may be delivered in separate batches; please refer to the actual received goods. Upon receipt, store components separately at the temperatures specified in the component list. The shelf‑life is 12 months from the date of manufacture. 2. * Sterile, ** Sterile and RNase‑free. |
Required Materials (Not Included in Kit)
Ⅰ Consumables & Supplies
1. Peeling knife, utility knife, glass cutting board and other tools for cutting plant materials into pieces suitable for juicing
2. 1‑2 beakers (400 mL)
3. Multiple 50 mL centrifuge tubes and 1.5 mL EP tubes
4. pH test strips: universal pH 1‑10; precision pH 5.5‑8.5
Ⅱ Instruments and Equipment
1. Electronic balance
2. Grinding‑type juicer (recommended; cold‑press juicer is preferred for purchase reference)
3. High‑speed centrifuge (4 °C compatible)
4. Water bath
5. Vortex mixer
Ⅲ Reagents
Sterile 1×PBS solution
Operating Procedure
Step 1: Pre‑juicing Preparation
1. Disinfect the juicer: Disassemble all juicer components, spray thoroughly with 75% ethanol, then place in a biosafety cabinet for UV disinfection for no less than 20 min.
2. Sample preparation: Prepare fresh plant samples according to kit throughput.
Note: The 2T kit processes 20 mL plant juice per run.
3. Sample treatment: Thoroughly wash the outer surface of fresh plant materials, wipe dry with lint‑free cloth, perform UV disinfection in a biosafety cabinet for no less than 20 min, then cut plants into small pieces suitable for juicing.
Notes:
a. For thick‑skinned plants (e.g., *Pueraria lobata*): wash outer surface, disinfect, peel off skin, then cut into small pieces or thin slices.
b. For thin‑skinned plants (e.g., notoginseng, rehmannia, turmeric): wash outer surface, disinfect, and cut directly into small pieces for juicing.
Step 2: Juicing
1. Assemble disinfected juicer components inside the biosafety cabinet following the manufacturer’s instructions.
2. Juicing: Feed pre‑cut plant pieces into the juicer. For hard‑textured plant materials, feed materials in small portions multiple times. Collect plant juice upon completion.
Note: Perform juicing inside a biosafety cabinet if low‑endotoxin preparations are required.
Step 3: Centrifugal Clarification
1. Low‑speed impurity removal: Centrifuge crude plant juice at 5 000 × g, 4 °C for 10 min, collect supernatant.
2. High‑speed impurity removal: Centrifuge the recovered supernatant at 10 000 × g, 4 °C for 10 min, collect supernatant.
Step 4: Vesicle Isolation
1. Supernatant pre‑treatment: Add well‑mixed Solution A to clarified plant juice. For every 20 mL clarified plant juice, add 200 μL Solution A; scale volumes proportionally for other sample inputs.
Note: Mix Solution A thoroughly before use.
2. Incubation after mixing: Secure tube caps, invert tubes 8‑10 times for mixing, then incubate the mixture in a 37 °C water bath for approximately 16 h (overnight).
3. pH adjustment: Remove incubated samples. Inside a biosafety cabinet, adjust the pH of the mixture to 7.0 using kit‑supplied 1 M NaOH and 1% citric acid.
4. Post‑pH‑adjustment high‑speed clarification: Centrifuge pH‑adjusted mixture at 10 000 × g, 4 °C for 20 min and collect supernatant.
5. Filtration: Sequentially filter supernatant through 1 μm filter membrane followed by 0.45 μm filter membrane.
Note: Retain only the 1 μm filtration step and skip 0.45 μm filtration if isolating plant extracellular vesicles ranging 30‑1000 nm.
6. Addition of isolation reagent: Add Solution B to the filtered supernatant. Add 5 mL Solution B for every 20 mL clarified plant juice; scale volumes proportionally for other sample inputs.
7. Second incubation: Tighten tube caps, vortex‑mix for 1 min, then incubate mixture statically at 4 °C for approximately 4 h.
8. Pelleting plant extracellular vesicles: Centrifuge incubated mixture at 10 000 × g, 4 °C for 60 min, discard supernatant. Re‑spin the same tubes at 10 000 × g, 4 °C for 2 min and aspirate residual supernatant completely.
Note: The pellet is enriched in plant extracellular vesicles; remove supernatant as completely as possible.
9. Vesicle resuspension: Gently pipette appropriate volume of 1×PBS to fully resuspend the pellet. Transfer resuspended suspension into a new 1.5 mL EP tube. Recommended volume: ~400 μL 1×PBS per 20 mL starting plant juice.
10. Harvest crude plant extracellular vesicles: Centrifuge the resuspended suspension in 1.5 mL EP tubes at 12 000 × g, 4 °C for 2 min. Retain supernatant, which contains enriched plant extracellular vesicle particles.
Note: If substantial pellet remains, repeat centrifugation at 12 000 × g for 2 min until no obvious pellet is observed; collect supernatant each time.
Step 5: Purification and Storage of Plant Extracellular Vesicles
1. Vesicle purification: Transfer crude plant extracellular vesicle preparation to the upper chamber of an exosome‑purification filter column (EPF column). Centrifuge at 3 000 × g, 4 °C for 10 min. Collect flow‑through at the bottom of EPF column, which represents purified plant extracellular vesicles.
Notes:
a. EPF columns are single‑use; do not reuse.
b. Omit this purification step if large‑size plant extracellular vesicles are desired.
2. Storage: Aliquot purified plant extracellular vesicles into appropriate volumes and store at −80 °C for downstream assays.
Precautions
1. Upon receipt, store Solution A (2T kit) at −80 °C immediately. Thaw at 4 °C before use and consume promptly after thawing. Avoid repeated freeze‑thaw cycles.
2. A brown‑yellow precipitate may form in Solution A upon standing; this is normal. Mix thoroughly prior to use.
3. For strict low‑endotoxin requirements, perform the juicing step strictly inside a biosafety cabinet, and ensure critical supplies and instruments are fully disinfected before use.
4. Store purified plant extracellular vesicles at −80 °C. Repeated freeze‑thaw cycles will cause vesicle rupture and should be avoided.
危险品化学品经营许可证（带存储）