Exosomes are small vesicles (30-150 nm in diameter) secreted by living cells, characterized by a typical lipid bilayer structure. They exist in cell culture supernatants, serum, plasma, saliva, urine, amniotic fluid, and other biological fluids. Exosomes carry various important biomolecules such as proteins, lipids, DNA, and RNA. They play a crucial role in intercellular material and information transfer and hold promise as early diagnostic biomarkers for various diseases. This kit employs a unique isolation technology to rapidly obtain a large quantity of intact exosome particles from cell culture supernatants. It is suitable for downstream applications including cell co-culture, electron microscopy analysis, Western Blot, quantitative PCR (qPCR), and high-throughput sequencing. The maximum processing volume is 200 mL of cell culture supernatant. Product Information Product Information. Catalog Number Size Hieff™ Quick Exosome Isolation Kit Plus(for Cell Culture Media) 41205ES02 2 T 41205ES10 10 T 41205ES20 20 T Components Component No. Component Name 41205ES02 41205ES10 41205ES20 41205-A Exosome Concentration Solution* 10 mL 25 mL 50 mL 41205-B Exosome Purification Filter* 2 Tubes 10 Tubes 20 Tubes [Note]:* Nuclease-free, Sterile Storage Transport at room temperature. Store at room temperature. Shelf life: 2 years. Instructions I. Sample Pre-processing 1. Sampling: Collect fresh samples and place them on ice for use. For frozen samples, thaw them in a 25°C water bath, then place them on ice after complete melting. 2. Initial Sample Volume: The recommended volume for a single extraction is no less than 20 mL of cell culture supernatant. 3. Remove Cell Debris: Transfer the sample to a centrifuge tube and centrifuge at 4°C, 3,000×g for 10 min to remove cell debris. (If there is a large amount of precipitate, repeat centrifugation at 3,000×g for 10 min until no obvious precipitate remains, collecting the supernatant each time). 4. Remove Impurity Debris: Transfer the supernatant to a new centrifuge tube and centrifuge at 4°C, 10,000×g for 10 min to remove impurity debris. 5. Supernatant Transfer: Transfer the supernatant (free of impurity debris) to a new centrifuge tube. II. Exosome Precipitation 1. Supernatant Pre-treatment: Add 41205-A Reagent to the clarified supernatant. The specific volume is as follows (adjust proportionally for other sample volumes): Sample Name Sample Volume 41205-A Reagent Volume Cell Culture Supernatant 20 mL 5 mL 2. Mixing: After adding the reagent, tighten the cap and mix thoroughly using a vortex mixer for 1 min. Incubate at 4°C for at least 8 hours (Note: Extending the incubation time can improve exosome yield, but do not exceed 24 hours). 3. Precipitate Exosomes: Centrifuge the mixture at 4°C, 10,000×g for 60 min. Discard the supernatant (Note: Aspirate as much supernatant as possible). 4. Secondary Centrifugation: Centrifuge the tube containing the pellet again at 4°C, 10,000×g for 2 min. Discard the supernatant (Note: Aspirate as much supernatant as possible). 5. Resuspend Pellet: Resuspend the pellet by pipetting up and down with an appropriate volume of 1×PBS. Transfer the resuspended solution to a new 1.5 mL centrifuge tube (Recommended: ~200 μL of 1×PBS for every 20 mL of cell culture supernatant). 6. Harvest Exosome Particles: Centrifuge the 1.5 mL tube at 4°C, 12,000×g for 2 min. Retain the supernatant, which is enriched with exosome particles (If there is significant precipitate, repeat centrifugation at 12,000×g for 2 min until no obvious precipitate remains, collecting the supernatant each time). III. Exosome Purification 1. Purify Exosomes: Transfer the crude exosome particles (supernatant from step 2.6) to the upper chamber of the 41205-B (EPF Column). Centrifuge at 4°C, 3,000×g for 10 min. Collect the liquid at the bottom of the collection tube; this liquid contains purified exosome particles (Note: The EPF Column is for single use only). 2. Storage: Aliquot the purified exosomes into suitable volumes and store at -80°C for subsequent experiments. Notes 1. To ensure the exosomes are derived from your cells, it is best to culture cells using exosome-depleted serum. 2. For further purification, affinity purification using antibody-coated magnetic beads is recommended. 3. This product is specifically designed for exosome isolation from cell culture supernatants. It is not suitable for exosome extraction from serum or plasma. For serum/plasma exosome isolation, please select the Serum/Plasma Exosome Rapid Extraction Kit. 4. For your safety and health, please wear a lab coat and disposable gloves when handling this product. 5. For research use only! Documents: Safety Data Sheet 41205_MSDS_HB260525 Manuals: 41205_Manual_Ver.EN20260515
