293T外泌体是293T无血清培养细胞上清液中分离提取获得的高纯度外泌体颗粒，直径在30-150 nm。293T细胞系属于稳定细胞系，对细胞培养要求较低，生长效率高，能高效地产生大量生产重组蛋白，可作为外泌体工程化内源性改造的重要媒介。
从293T细胞分离的外泌体可用于透射电镜分析、NTA粒径分析、核酸蛋白分析、细胞学及动物学实验等。
产品优势：
严格的质控标准：多项质控分析，有严格的浓度、纯度等不同质控标准。
完整的表征结果：外泌体原料产品按照MISEV2018指南，从电镜形态、粒径大小及颗粒分布、WB三阳一阴蛋白标志物三个维度进行表征。
广泛的应用场景：可用于实验流程及功能实验的对照；可用于靶向改造、装载分子等工程化改造，以及外泌体治疗产品的开发。
产品规格：
名称
| 人胚肾细胞（293T）外泌体
|
规格
| 1E+10 Particles/瓶
|
外观
| 白色粉末
|
pH
| 7.0 |
无菌检测
| 无菌生长
|
总蛋白浓度
| （BCA）
|
纯度
| ≥90% |
案例展示：
1. NTA检测外泌体的粒径分布和颗粒浓度：
将得到的外泌体震荡分散均匀，用PBS稀释至合适倍数，标记好样本名称、稀释倍数、完成样本的稀释配制；测样之前，先测试稀释液：移液枪吸取200μL稀释液上机检测，确认组件、仪器
都处于正常运行；稀释液测量完成，测试结果正常后开始测试样本，取稀释后的200μL样本上机检测；待计数颗粒数达到100个以上时停止测试，导出数据结果，完成样本检测。
2. TEM观察外泌体的形态：
重悬外泌体至50-100μL 2% PFA中，将5μL混合悬液加到Formvar carbon载样铜网上；也可将5-10μL混合悬液滴加到封口膜上，将铜网Formvar膜面朝下放在悬液上。每个样品准备2-3个铜网；将100μL PBS 加到封口膜上。用镊子将铜网 (Formvar 膜面朝下) 放在PBS液滴上清洗 (在所有步骤中，都应保持Formvar膜面湿润，而另一面干燥)；将铜网放在50μL 1%戊二醛液滴上5 min；将铜网放在100μL ddH₂O洗涤8次，每次洗2min；将铜网放在50μL草酸双氧铀液滴上 (pH 7.0)，5min；将铜网放在50甲基纤维素液滴上10min，冰上操作；铜网放到样品台顶端的不锈钢环上，用滤纸吸去多余液体；空气中干燥5-10min；将铜网放在样品盒内，80kV下拍摄电镜照片。
3. Western Blot 检测外泌体标志物 (三阳一阴)：
将分离纯化得到的外泌体加入裂解液 (E778170)，裂解后吸取上清液，根据测定蛋白浓度稀释样本至合适浓度；裂解液加入 4×LDS上样缓冲液 (T466588)，95°C煮5min，待降至室温后，瞬离；加样：将15μL样本全部加到泳道中，并在样本首尾加上marker；电泳：190V，70min；提前10min用甲醇活化PVDF膜；转膜：转膜液需要提前预冷，275mA，70min；封闭：配置1% BSA的TBST溶液，室温封闭1h；孵育一抗：4°C，摇床上过夜，抗体用1% BSA的TBST溶液稀释；洗膜：1xTBST 洗三次，10 min/次；孵育二抗：抗体用1% BSA的TBST溶液稀释，室温摇床1h；洗膜：1xTBST洗三次，10min/次；曝光拍照。
注意事项和免责声明：
本品仅限于专业人员的科学研究使用，不得用于临床诊断或治疗，不得用于食品或药品。
293T exosomes are high-purity exosome particles isolated and extracted from the supernatant of 293T cells cultured in serum-free medium, with a diameter ranging from 30 to 150 nm. The 293T cell line is a stable cell line with low requirements for cell culture, high growth efficiency, and the ability to efficiently produce large quantities of recombinant proteins, thus serving as an important medium for engineering and endogenous modification of exosomes.
Exosomes isolated from 293T cells can be used in transmission electron microscopy analysis, NTA particle size analysis, nucleic acid and protein analysis, cytological and zoological experiments, etc.
Product Advantages:
Strict quality control standards: Multiple quality control analyses are conducted, with strict standards for various aspects such as concentration and purity.
Comprehensive characterization results: Exosome raw material products are characterized in three dimensions in accordance with the MISEV2018 guidelines, including electron microscope morphology, particle size and particle distribution, and WB three-positive and one-negative protein markers.
Wide range of application scenarios: It can be used as a control in experimental procedures and functional experiments; it can be applied in engineering modifications such as targeted modification and molecular loading, as well as the development of exosome therapeutic products.
Product Specifications:
Name
| 239T Exosomes
|
Specifications
| 1E+10 Particles/EA
|
Appearance
| White Powder
|
pH
| 7.0 |
Aseptic Testing
| Aseptic
|
Total Protein Concentration
| （BCA）
|
Purity
| ≥90% |
Case Presentation:
1. NTA detection of exosome particle size distribution and particle concentration:
Shake and disperse the obtained exosomes evenly, dilute them to an appropriate multiple with PBS, mark the sample name and dilution multiple, and complete the dilution preparation of the sample; before testing the sample, test the diluent first: absorb 200μL of diluent with a pipette for on-machine detection, and confirm that the components and instruments are in normal operation; after the measurement of the diluent is completed and the test result is normal, start testing the sample, take 200μL of the diluted sample for on-machine detection; stop the test when the number of counted particles reaches more than 100, export the data results, and complete the sample detection.
2. Observation of exosome morphology by TEM:
Resuspend exosomes in 50-100μL of 2% PFA. Add 5μL of the mixed suspension to a Formvar carbon-coated copper grid; alternatively, drop 5-10μL of the mixed suspension onto a piece of parafilm and place the copper grid with the Formvar film facing down on the suspension. Prepare 2-3 copper grids for each sample. Add 100μL of PBS onto the parafilm. Use tweezers to place the copper grid (with the Formvar film facing down) on the PBS droplets for washing (during all steps, keep the Formvar film surface moist while the other surface remains dry). Place the copper grid on 50μL of 1% glutaraldehyde droplets for 5 minutes. Wash the copper grid on 100μL of ddH₂O 8 times, 2 minutes each time. Place the copper grid on 50μL of uranyl oxalate droplets (pH 7.0) for 5 minutes. Place the copper grid on 50μL of methyl cellulose droplets for 10 minutes, operating on ice. Put the copper grid on the stainless steel ring at the top of the sample stage and blot off excess liquid with filter paper. Air-dry for 5-10 minutes. Place the copper grid in the sample box and take electron microscope photos at 80kV.
3. Western Blot Detection of Exosome Markers (Three Positive and One Negative):
Add the isolated and purified exosomes to the lysis buffer (E778170). After lysis, aspirate the supernatant and dilute the sample to an appropriate concentration according to the measured protein concentration. Add 4×LDS loading buffer (T466588) to the lysis buffer, boil at 95°C for 5 minutes, and perform a quick centrifugation after cooling to room temperature. Loading: Load all 15μL of the sample into the lane, and add markers at the start and end of the sample lanes. Electrophoresis: 190V for 70 minutes. Activate the PVDF membrane with methanol 10 minutes in advance. Membrane transfer: The transfer buffer needs to be pre-cooled in advance, 275mA for 70 minutes. Blocking: Prepare 1% BSA in TBST solution and block at room temperature for 1 hour. Primary antibody incubation: Incubate overnight at 4°C on a shaker; dilute the antibody with 1% BSA in TBST solution. Membrane washing: Wash three times with 1×TBST, 10 minutes each time. Secondary antibody incubation: Dilute the antibody with 1% BSA in TBST solution, and incubate on a shaker at room temperature for 1 hour. Membrane washing: Wash three times with 1×TBST, 10 minutes each time. Exposure and photography.
Precautions and Disclaimer:
This product is limited to scientific research use by professional personnel. It must not be used for clinical diagnosis or treatment, nor for food or drugs.
ZJ25F0926761
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