Description
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:Name239T ExosomesSpecifications1E+10 Particles/EAAppearanceWhite PowderpH7.0Aseptic TestingAsepticTotal 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