0.22 μm filter

OMVs were extracted by an electrophoresis and dialysis-based method (ELD) with a 300 kDa cut-off dialysis bag [17 (link)]. Specially, 200 ml of DMEM (Gibco, Shanghai, China) inoculated with the single colony was incubated at 37 ℃ with shaking at 200 rpm for 20–24 h. The culture solution was centrifuged at 10,000×g for 10 min to collect the culture supernatant, which was then filtered through a 0.22 μm filter (Beyotime, Shanghai, China) to remove bacteria and bacterial debris. Next, the OMVs of CRKP was isolated from filtered supernatant as previously described [17 (link)].
The OMVs of CRKP were photographed by transmission electron microscopy (TEM). Five µL of OMV suspension fixed with anhydrous ethanol was dropped onto a 200-mesh copper wire at room temperature for 1 min. Then, the sample was drained with filter paper and negatively dyed with 2% uranium acetate for 1 min. Afterwards, the excess dye was drained with filter paper. Then, the OMVs were observed by a Tecnai G2 Spirit Bio transmission electron microscope (FEI, America). NanoSight NS500 (Thermo Scientific, America) was used for determination of OMVs, and the particle size and concentration of OMVs were analyzed by NTA software.

Fresh human CRC and adjacent normal tissue samples were obtained directly from the operating room. Tissues were weighed and preserved in Falcon tubes containing 10 mL of DMEM with antibiotics (100 U/mL penicillin, and 100 mg/mL streptomycin) to avoid bacterial or fungal contamination. Tissues were cut into similar sized pieces and cultured with DMEM for 12 h. Then, collected conditioned medium (CM) was collected, and filtered through 40-μm cell strainers (Thermo Fisher Scientific, MA, USA) to obtain tissue-conditioned medium and stored at − 80 °C until further use.
After the SW480 cells (vector/WNT4-HA) grew to 70–80% confluency, then cells cultured in DMEM medium (no FBS) for 24 h. CM was obtained and centrifuged (1000 rpm, 10 min), and the supernatant was filtered through a 0.22 μm filter (Beyotime Biotechnology, China) and stored at 4 °C for treating HUVECs.