0.22 μm filter

SCFA concentration of luminal content was measured following the previous protocol [36 ]. Briefly, samples were thawed on ice and approximately 0.5 g sample was added to 8 mL of deionized water. Then, the mixture was thoroughly homogenized by vertexing for 1 min and centrifuged at 13,000 × g for 5 min. The supernatant was diluted 50 times and filtered through a 0.22-μm filter (Millipore, Bedford, OH). Extracted sample solution (25 μL) was analyzed by a high-performance ion chromatography of ICS-3000 (Dionex, USA).
To determine the SCFA concentration of the bacteria medium, 1 mL medium was collected at each time point and centrifuged at 13,000 × g for 5 min. The supernatant was diluted 100 times and filtered through a 0.22-μm filter (Millipore, Bedford, OH). Other procedures were the same as above.

The CD3 and EpCAM fragments were purified by Capto L chromatography (Cytiva, Uppsala, Sweden) through the Akta150 system (Cytiva, Uppsala, Sweden). The supernatant was centrifuged and flirted through a 0.45 μm filter membrane. The column was loaded with binding buffer (20 mM sodium phosphate, 150 mM NaCl, pH 7.4), supernatants, wash buffer (100 mM sodium citrate, pH 5.0), and eluted with elution buffer (100 mM sodium citrate, pH 2.8). The elution was neutralized to pH 7.0 with Tris-HCl buffer (1 M, pH 8.0). Fragment A and B samples were dialyzed into phosphate buffer (PBS, pH 7.4) followed by splicing reaction catalyzed mediated by split intein.
After dialyzing all of the fragments in PBS buffer and adding 2 mM dithiothreitol (DTT, Sigma Aldrich, Shanghai, China), 100 mM fragment A was combined with 375 mM fragment B and incubated at 37°C for 2 h. The mixture was dialyzed to PBS to remove DTT, sterilized using a 0.22 μm filter (Millipore, Shanghai, China), and the oxidation reaction was carried out at room temperature for 2–3 days. The CD3×EpCAM BsAb was isolated through Protein A affinity chromatography (Cytiva, Uppsala, Sweden). The eluates were adjusted to pH 7.0 using Tris-HCl buffer, dialyzed to PBS, and sterilized through a 0.22 μm filter (Millipore, Shanghai, China).

Blood samples from heathy Holstein cows during early pregnancy (G.D. 60, n = 5) and non-pregnancy (n = 5) were sampled by coccygeal venipuncture in accordance with protocols approved by the Huazhong Agricultural University Animal Care and Use Committee (Wuhan, China) and collected in evacuated blood tubes containing dipotassium ethylenediaminetetraacetic acid (K₂EDTA) as an anticoagulant. The samples were immediately placed in ice water and centrifuged within 30 min at 1500×g for 12 min at 4 °C. Plasma exosomes were isolated and characterized as previously described⁶⁶ . In brief, plasma (2 mL) was diluted with an equal volume of 1× phosphate-buffered saline (PBS) (pH 7.4) and centrifuged at 2000×g for 30 min at 4 °C. The supernatant was then centrifuged at 12,000×g for 45 min at 4 °C. The resultant supernatant fluid (4 mL) was filtered through a 0.22-μm filter (Millipore, USA), transferred to an ultracentrifuge tube, and centrifuged at 100,000×g for 75 min at 4 °C (Beckman Optima XE-90, SW32 Ti rotor, Beckman Coulter, Inc. USA). The pellet was suspended in PBS, filtered through a 0.22-μm filter (Millipore, USA) and centrifuged at 100,000×g for 2 h at 4 °C. Finally, the pellet containing the enriched exosome population was resuspended in 50 μL PBS (HyClone, USA) and stored at −80 °C.

Mouse. TES were prepared from excised non-ulcerated EL4, LLC, MC38, Ret melanoma, or KPC pancreas tumors. A small tumor piece (0.5 g) was harvested, minced into pieces <3 mm in diameter and resuspended in complete RPMI. After 16–18 h of incubation at 37 °C with 5% CO₂, the cell-free supernatant was collected using 0.22 μm filters (EMD Millipore) and kept at −80 °C.
Human. TES was prepared from surgically removed tumors and a small tumor piece (0.1–0.5 g) was processed. After 16–18 h of incubation at 37 °C with 5% CO₂, the cell-free supernatant was collected using 0.22 μm filters (EMD Millipore) and kept at −80 °C.

The cells were seeded into T175 flasks (Thermo Fisher Scientific). At 80% confluence, the cells were washed three times with PBS (Gibco by Life Technologies), the medium was changed to serum‐free medium, and the cells were further incubated for 24 h. SW480_EV‐S and SW480_EV‐M were isolated from the conditioned media by sequential centrifugation and ultrafiltration. Briefly, cell debris and floating cells were removed by centrifugation at 300 g for 10 min and 4500 g for 5 min, and the supernatant was stored at −80 °C. The supernatant was thawed at 37 °C, and medium EVs were isolated by centrifugation at 17 000 g (11 930 r.p.m., k‐factor 2112) for 30 min using a fixed‐angle Sorvall SS‐34 rotor (Kendro Laboratory Products, Newtown, CT, USA). The pellet was resolved in serum‐free medium and concentrated using Amicon Ultra‐4100 kDa Centrifugal Filter Devices (Merck Millipore, Cork, Ireland). Prior to small EV isolation, to exclude particles larger than 220 nm, the 17 000 g supernatant was gently filtered through 0.22‐μm filter (Merk Millipore). Small EVs were isolated by Centricon‐70 Plus 100 kDa Centrifugal Filter Columns (Merk Millipore) and centrifuged at 3500 g for 15 min with a washing step in PBS at 3500 g for 10 min. All centrifugations were performed at room temperature. All samples were stored at −80 °C.

Roots, stems, leaves and spikes of two-year-old P. vulgaris were ground in liquid nitrogen. The ground samples (0.5 g) were dissolved in 10 ml of 80% ethanol and sonicated for 60 min. The extracts were collected by centrifugation and filtered using a 0.22 μm filter (Merk Millipore, USA). UPLC and LC-MS/MS analyses were performed using the ACQUITY UPLC I-Class system (Waters) as described as in vitro enzymatic activity assay of recombinant proteins. Three biological and three technological replicates were carried out for analysis of each tissue.

Zoledronate (ZOL, Sigma-Aldrich, Germany) was dissolved in sterile phosphate-buffered saline (PBS, Gibco, USA) with a stock solution at 100 mM and filter sterilized using a 0.22 μm filter (Merk Millipore, USA). ZOL solutions were stored at 4°C.