Phosphate buffered saline (pbs)

B. tabaci adults (acquired ToCV for 48 h) were immediately placed into the fixed fluid after 12 h. The B. tabaci was dehydrated by gradient alcohol and paraffin-embedded. The paraffin was sliced, and slices were dewaxed and dehydrated. Proteinase K (20 μg/mL) working solution (Servicebio, Wuhan, China) was added to cover objectives, incubated at 37 °C for 15 min, then washed three times (5 min each) in PBS (pH 7.4) (Servicebio, Wuhan, China). After washing in PBS for 15 min, the slices were rinsed in hybridization buffer (20 mM Tris-HCl, pH 8.0, 0.9 M NaCl, 0.01% (wt./vol) sodium dodecyl sulfate, 30% (vol/vol) formamide) (Servicebio, Wuhan, China) for the pre-hybrid (without the probe). Then, 1 μmol of the fluorescent BtPPO1 probe (conjugated with Cy3) was added into the slices. The slices were rinsed in hybridization buffer again. After that, 1 μmol of the fluorescent ToCV probe (conjugated with FAM) was added into the slices. Then, the hybridized slices were rinsed three times in hybridization buffer and incubated with DAPI (Servicebio, Wuhan, China) for 8 min in the dark, then mounted before taking photos with a positive fluorescence microscope (Nikon, Tokyo, Japan). Probe sequences are listed in Table S1.

To isolate EVs from cultured cell lines, the cells were initially rinsed with phosphate-buffered saline (PBS) (Servicebio, #G4202, China) prior to incubation in culture media devoid of EVs. After 3–4 days, the cultured supernatant was collected for further extracting EVs. To separate plasma, whole blood collected in EDTA was centrifugated at 1500g for 10 min at 4 ℃. Tissue explant culture followed a previously described method [41 (link)].
The conditioned medium of either cultured cells, tissue explants, or plasma underwent a series of centrifugations: first at 500g for 10 min, then 3000g for 20 min, and finally 12,000g for 20 min, all at 4 ℃. EVs from the above sources were then obtained by ultracentrifugation at 100,000g for 70 min at 4 ℃, followed by a wash in PBS (Servicebio, #G4202, China) and re-ultracentrifugation. The concentration of EVs was determined using BCA protein assay (Thermo Fisher Scientific, #23225, USA).

This method has been reported in our recent study [2 (link)]. Briefly, the collected intervertebral disc tissue was transported to laboratory using 0.9% sterile saline within two hours. Then, the tissue was washed 3 times using PBS (Servicebio, Wuhan, China) in super clean bench. Next, gel-like NP tissue was separated and digested using 0.25% Trypsin-EDTA (C0209-100 mL, Beyotime, Shanghai, China) for 30 minutes and 0.2% collagenase type II (Invitrogen, USA) for another 1 h at 37°C under a shaker (70 r/min). Finally, the isolated NP cells were resuspended in complete culture medium (Gibco; Thermo Fisher Scientific, Inc.) and cultured in a 37°C incubator. At the third, half of the culture medium will be replaced by complete medium. Five days after isolation, the spindle-shaped NP cells will move out and adhered at the bottom which we called passage 0. When the cell density reaches about 80%, the NP cells could be used further experiments.

The UCMSCs with a confluence of 90–95% were washed twice with 1 X phosphate buffered saline (PBS, Servicebio, China), added with α-MEM containing different concentrations of staurosporine (STS, Enzo Life Sciences, USA) (250, 500, or 750 nM), and incubated at 37 °C for 12 h in 5% CO₂, or added with α-MEM containing 500 nM STS and incubated for different time (4, 8, 12, or 16 h). ApoVs were isolated from the medium of apoptotic MSCs by sequential centrifugation (800g for 10 min, 2000g for 10 min, and 16,000g for 30 min) at 4 °C as we reported previously [67 (link)]. Finally, the pellet was washed once with 0.22 μm-filtered PBS to purify apoVs.

Pancreatic carcinoma xenografts were established according to a previously described procedure.^[30] Briefly, the human pancreatic carcinoma cell line PANC‐1 (5 × 10⁵ cells) suspended in 200 µL phosphate‐buffer saline (PBS, Servicebio, Wuhan, China) was subcutaneously inoculated in the flank of male BALB/c‐Nu mice. When tumors grew to ≈100 mm³, tumor‐bearing mice were randomly divided into three groups, including negative control group without any treatment, a control group with SMF treatment and therapy group with EYF treatment (n = 6). At the end of the experiment, the tumors were removed, weighed, photographed, and subjected to hematoxylin and eosin (H&E) staining and immunohistochemical analysis.