0.22 μm syringe filter

The fresh fecal samples were collected at the 27th week and stored at −80 °C until analysis. The fecal samples were prepared by mixed with methanol at a ratio of 3 mL/g [37 (link)] and homogenized (8 m/s, 15 s) by adding ceramic beads (1 mm, Omni International, Bedford, NH, USA). The mixtures were centrifuged (12,000 rpm, 10 min, 4 °C) (Eppendorf, Hamburg, Germany), and the supernatants were filtered using 0.22 μm syringe filters (Millipore Corp, Billerica, MA, USA). The serum samples were extracted by mixing with acetonitrile at a ratio of 1:3 and vortexed immediately to remove protein [38 (link)]. The mixtures were then centrifuged (12,000 rpm, 10 min, 4 °C) to obtain the supernatants for processing. The procedural blank sample of water was used to monitor the contamination during sample preparation. The quality control (QC) samples of all mixing extraction were processed before and during sample processing to ensure the quality of metabolic profiling [38 (link)].

HUVECs provided by the China Center for Type Culture Collection (CCTCC, China) underwent culture in F12-K basal medium supplemented with 10% FBS, ECGS and 0.1 mg/ml heparin sodium at 37 °C under 5% CO₂. UA (Sigma, USA) was dissolved in serum-free F12-K medium, incubated at 37 °C with constant shaking, and filtered using 0.22-μm syringe filters (Millipore, USA) [11 (link)]. The UA concentration did not decrease when the urate-medium was stored at 4 °C within 72 h. Therefore, only the urate-medium prepared within 72 h was used. A polarizing microscope (Olympus, Japan) detected no UA crystals during cell treatments.

Passage 5–6 human bone marrow-derived MSCs from a 22-week-old foetal donor (3H Biomedical, Sweden) were used for all experiments. MSCs were cultured as a monolayer in tissue culture flasks (Corning, UK) in MesenPRO RS medium (Invitrogen, UK) supplemented with 1% penicillin/streptomycin and 2 mM glutamine. The growth medium was changed every 4–5 days until the cells were 70–80% confluent. MSCs were then dissociated with 0.5% trypsin-EDTA (Sigma-Aldrich, UK) and counted. For IL-1α-primed CM (αCM) preparation, MSCs were seeded in 6-well plates (Corning, UK) at a density of 1.75 × 10⁵ cells/well and incubated for 24 h. MSCs were then treated with 10 ng/ml human recombinant IL-1α (R&D Systems, UK) for 5 min. Cells were washed twice with PBS then serum-free MesenPRO RS medium (without supplement) was added. After 24 h, αCM was collected, cell debris was removed using 0.22-μM syringe filters (Millipore, UK) and 10× concentrated using 3000 MWCO Vivaspin centrifugal concentrators (Generon, UK) according to the manufacturer’s instructions. For the vehicle, serum-free MesenPRO RS medium was also concentrated 10×. For all in vivo experiments, 400-μl conditioned treatments derived from 3.5 × 10⁵ cells were prepared in advance and stored at − 80 °C.

For the EDs isolation, nomenclature and functional characterization, we tried to follow the minimal information for studies of extracellular vesicles 2018 (MISEV2018) guidelines^{29 (link)}. However, we still have issues in removing various kinds of lipoproteins^{27 (link)} and albumin, so we used extracellular derivatives (EDs) instead of using extracellular vesicles EVs. Total EDs were isolated and purified from 200 mL of serum using the miRCURY Kit (Cat. 76603, Qiagen, CA) according to the manufacturer’s instructions, with minor modification. Briefly, using 0.22 μm syringe filters (Millipore, MA), serum samples were filtered to avoid larger particles. Pre-filtered samples were then mixed with Precipitation Buffer A for 12 hours and centrifuged at 1500 × g for 30 minutes. The precipitated EDs were eluted with filtered 1x PBS (100 μl) and used for further analysis. The remaining samples were stored at −20 °C for less than 10 days.

Culture supernatants or freshly-thawed plasma were centrifuged at 2,000 × g for 10 min at room temperature (RT) and at 10,000 × g for 30 min at 4°C followed by filtration on 0.22 μm syringe-filters (Millipore). Pre-conditioned supernatants were concentrated from 50 to 1 mL on Vivacell 100 filter units (MWCO 100,000, Sartorius Corp, Bohemia, NY, USA). Aliquots (1 mL) of pre-conditioned plasma or concentrated supernatants were loaded on mini-SEC columns (18 (link)), and exosomes were eluted with PBS. Exosomes were collected in the void volume fraction #4 (1 mL). For some experiments, particularly for Western blots, #4 miniSEC fractions were concentrated using 100,000 MWCO Vivaspin 500 Centrifugal Concentrators (Sartorius Corp) by centrifugation at 2,000 × g for 10–15 min.