Exo spin midi columns

Plasma samples from TSC patients with a TSC2 mutation were obtained from the TSC Alliance Biosample Repository (Van Andel Institute, Michigan, USA). Age and sex matched healthy donor plasma samples were obtained from the Cardiff University Biobank (University Hospital of Wales, Application Number 21‐0002). Plasma EVs were isolated using Exo‐spin™ midi columns (CELL Guidance Systems), and assessed for CD9 and CD63 expression using TRIFic™ detection assays (CELL Guidance Systems), using established protocols similar to as published (Welton et al., 2015 (link)).

Stromal cell‐conditioned media was collected every five days, and EVs purified by filtration and ultracentrifugation, using established methods (Thery et al., 2006 ). Briefly, conditioned media were centrifuged twice at 400 × g for 6 m, then once at 2000 × g for 15 m, and filtered using a 0.2 µm syringe filter, to remove cells and debris. Supernatants were then centrifuged at 100,000 × g (max) (using Quick Seal tubes; 70 Ti rotor) for 2 h to pellet EVs, followed by a PBS wash at 100,000 × g (max) (using Quick Seal tubes; 70 Ti rotor) for 2 h. Isolation of EVs from cell‐conditioned media or patient serum (1 ml) by size exclusion chromatography was achieved using Exo‐spin™ midi columns (Cell Guidance Systems, Cambridge UK) (Welton et al., 2015 (link)). EV‐containing fractions were then pooled, and EVs isolated by centrifugation at 100,000 × g (using Quick Seal tubes; TLA‐110 fixed angle rotor, κ‐factor of 13, Optima Max‐XP ultracentrifuge; Beckman Coulter) for 2 h. EVs were resuspended in PBS, quantified using the micro‐BCA protein assay (ThermoFisher Scientific), and stored at –80°C.