Description
The exoEasy Maxi Kit uses membrane affinity spin columns to efficiently isolate exosomes and other extracellular vesicles from serum, plasma, cell culture supernatant and other biological fluids. The maxi column format allows the use of large sample volumes, including up to 4 ml plasma or serum or up to 32 ml cell culture supernatant. The procedure is very fast, consistent and highly suited for isolation of EVs for functional analysis of exosomes and other extracellular vesicles.
The exoEasy protocol for purification of exosomes and other extracellular vesicles is not only faster than ultracentrifugation, but also yields a cleaner preparation. Scanning electron microscopy shows that while both techniques deliver intact vesicles of the expected size, the preparation from ultracentrifugation also contains many smaller structures that do not match the expected size or shape for EVs (see figure “ Intact vesicles are eluted from the exoEasy membrane with higher purity compared with ultracentrifugation ”). Fully intact, spherical vesicles of different sizes between approximately 50-300 nm diameter can also be observed using cryo-electron microscopy. (see figure "Visualization of EVs isolated using the exoEasy Kit by cryo-electron microscopy"). Isolated vesicles are fully functional and can be used for several downstream applications including cell culture work (see figure “ Extracellular vesicles isolated using the exoEasy Kit are taken up efficiently by target cells (customer data) ”). Quality and quantity of eluted vesicles can be determined by a range of different methods. These include electron microscopy, physical characterization (e.g., Nanoparticle Tracking Analysis (NTA), or Tunable Resistive Pulse Sensing (TRPS)) and analysis of molecular constituents of vesicles, such as proteins, nucleic acids or lipids. Since particle counting techniques like NTA or TRPS do not distinguish vesicles from other particulate matter, particle counts performed in unprocessed biological fluids or crude preparations that still contain other particulate matter (e.g., protein complexes) may vastly overestimate the number of vesicles present. To standardize protocols and reporting in the EV field, the International Society of Exosomes has developed the Minimal Information for Studies of Extracellular Vesicles (MISEV) guidelines. These guidelines provide examples of EV-enriched protein markers that can be used to characterize EVs. However, analyzing protein expression in intact EVs can be challenging due to the limited amount of material and low expression levels of many EV proteins. In partnership with ProteinSimple (a Bio-Techne brand) QIAGEN has developed a standardized workflow for EV protein characterization. This new workflow combines a simple method for isolating EVs with automated protein separation and immunodetection of MISEV-recommended proteins. Following isolation with the exoEasy Maxi Kit, protein expression of intact EVs can be characterized on Simple Western by ProteinSimple. Simple Western is a western blotting platform for automated protein separation and immunodetection. Signals from potentially contaminating proteins such as APOA1 are efficiently depleted during isolation. (See figure “ EV protein analysis across plasma fractions using Simple Western automated western blot ”).
The exoEasy Kit improves upon traditional ultracentrifugation exosome or microvesicle isolation methods, yielding purified extracellular vesicles in just 25 minutes. The exoEasy Maxi Kit uses a membrane-based affinity binding step to isolate exosomes and other EVs from serum and plasma or cell culture supernatant. The method does not distinguish EVs by size or cellular origin, and is not dependent on the presence of a particular epitope. Instead, it makes use of a generic, biochemical feature of vesicles to recover the entire spectrum of extracellular vesicles present in a sample (see figure “ Characterization of extracellular vesicles isolated using the exoEasy Maxi Kit by NTA ”). It is therefore essential to completely remove cells, cell fragments, apoptotic bodies, etc., by centrifugation or filtration of samples before starting the protocol. The technology developed with Exosome Diagnostics, Inc., uses a spin column format and specialized buffers to purify exosomes from pre-filtered biological fluids; up to 4 ml using plasma or serum. For cell culture supernatants, processing of up to 32 ml sample (4 column loading steps) has been successfully tested. However, the concentration of vesicles in supernatants depends strongly on the cell type and culture conditions; therefore, we recommend starting with no more than 16 ml of supernatant for sample material that has not been tested with the kit previously. Higher sample volumes may result in reduced recovery of vesicles. The procedure is very fast, consistent and highly suited for functional analysis of exosomes and other extracellular vesicles. Particulate matter other than vesicles, such as larger protein complexes that are especially abundant in plasma and serum, is largely removed during the binding and wash steps.
The total procedure for isolating extracellular vesicles takes just 25 minutes. Pre-filtered plasma, serum or cell culture supernatant (with particles larger than 0.8 μm excluded) are mixed with Buffer XBP and bound to an exoEasy membrane affinity spin column. The bound exosomes are washed with Buffer XWP, eluted with 400 µl Buffer XE (an aqueous buffer containing primarily inorganic salts) and are then ready to use in physical or biochemical analysis. For certain applications, such as biological assays, an additional concentrating or buffer exchange step may be required. This can be achieved using ultrafiltration with a pore size of 100 kDa or smaller.
