Size exclusion chromatography (SEC) is a chromatographic separation technique were molecules and particles are separated based on size. As stationary phase sepharose beads are used, which are spheres with a diameter between 60 to 200 µm.
These beads contain pores through which molecules can penetrate in function of their size. Small molecules will elute later because they can penetrate through several pores. This in contrast to the molecules with a bigger size that can not penetrate easily through the pores. In the LECR, we make in-house SEC columns by using 10 mL syringes loaded with Sepharose CL-2B. These are used for EV isolation, separating EVs from the bulk of proteins present in the biological matrix.
Density gradient ultracentrifugation (UC) is used for the isolation and purification of extracellular vesicles (EV) from cell culture media and biological fluids. Density gradient separation involves the centrifugation of a biological sample through a centrifugation medium of higher or graded density. When exposed to an elevated centrifugal force, sample components migrate through the medium and separate based on their buoyant density. This results in the purification of EV populations through separation from contaminating protein and/or protein-RNA complexes.
Sucrose is a commonly used density centrifugation medium, but has a high viscosity and is hypertonic, making it less ideal for the separation of osmotically-sensitive materials. Therefore, at LECR, iodixanol (OptiprepTM)-based gradients are performed, since iodixanol is capable of forming iso-osmotic solutions at all densities. As such, it will not osmotically stress biological sample components.
Nanoparticle tracking analysis (NTA) is a technique routinely used for the quantification and characterization of extracellular vesicles. It uses light scattering and Brownian motion properties of particles in a sample to calculate the size distribution and concentration thereof. A laser beam is passed through a chamber containing particles in suspension and the resulting scattered light is detected, through a microscope (x20), with a video camera mounted on top. This camera then captures video files of the particles moving under Brownian motion and the NTA software then tracks each particle individually and calculates their hydrodynamic diameter using the Stokes-Einstein equation. The particle concentration is calculated based on the number of particles detected per screen and the total volume of the chamber. LECR is equipped with an LM10 NTA device provided with a 405nm laser for routine light scatter analysis and a 488nm laser for the analysis of green fluorescently labeled particles. For the latter a 500nm long-pass filter is placed in front of the camera and a continuous pump system is used for avoiding photo-bleaching effects. Furthermore the LM10 NTA device is provided with a 532nm laser.
Biomek 4000 automated workstation is a compact liquid handler that can assist in daily pipetting routines working from single vials or 96 well plates. It has 12 deck positions and can pipet from 1µL up to 1000µL. The project set-up is fully customizable and easy to use, fitting the needs for every type of experiment (sample aliquoting, ELISA's, MTT assays, cell staining,...). The biomek 4000 in LECR is equipped with a Positive Pressure HEPA Enclosure, which makes it possible to work in sterile conditions. In LECR the Biomek 4000 is especially used for the generation of iodixanol density gradients (see earlier), sample loading and fractionation of density gradients in order to ensure pure and reproducible isolation of extracellular vesicles from all kinds of samples.
