Aernoud Fiolet

272 Chapter 11 enrollment in the study. The study was conducted according to the Declaration of Helsinki. The data that support the findings of this study are available from the corresponding author upon reasonable request. Laboratory assessment Blood samples were drawn in participants one year after randomized treatment with colchicine or placebo. All samples were centrifuged at 1500 x g at 4°C for 15 minutes, and serum was stored at -80°C. Isolation of extracellular subfractions SerumEVor cell culturemediumEVsubfractionswere isolatedusing a standardized protocol. 21,22 For the total extracellular vesicle (TEX) subfraction 25µL of serum was diluted in 95µL PBS and 5µL Nano-mag®-D PEG-OH (1:25) (Micromod). The serum EV’s were then precipitated using Xtractt buffer (1:4) (Cavadis BV). Characterization of extracellular vesicles Iodixanol density gradient centrifugation of the subfraction was used to determine in which density fraction NLRP3 protein was present in the TEX fraction. Characterization of the density fractions of TEX isolation was described in detail before. 21,22 In these previous studies we show that with the precipitation method we used, that after density gradient centrifugation there was CD9 positivity (western blot) in the 1,05-1,09 density. In these densities also visible bilayer vesicles (electron microscopy) were present. In these density fractions, also NLRP3 proteinwas measured. This reveals the presence of EVs with NLRP3. In the current study all TEX density gradient fractions were characterized by CD9 western blot analysis with CD9 as EV protein marker. To get easy access to the overall data on characterization after density gradient an EV-track ID was created: EV200044. 22 Western Blot Western blot analysis was performed on a 4-12% gradient Bis/Tris gel (NuPage, Invitrogen). After blotting, the blot was incubated with the following antibodies: 1. Primary antibody: CD9 antibody (purified Mouse anti-CD9 (MAB25292) (R&D systems). 2. Secondary antibody: GOAT anti-mouse-HRP 1/1000) (P0447) (DAKO)