Inverted optical microscope

Galactosidase activity was quantified in intestinal and colonic organoids using the Senescence Cells Histochemical Staining kit (Sigma Aldrich) according to manufacturer’s instructions. Staining was developed over 10h and visualized in an inverted optical microscope (Leica). For quantitation, intestinal or colonic organoids were lysed in RIPA buffer (ThermoFisher) for 10min and centrifuged at 10,000×g for 5min, the supernatant was collected and the absorbance was measured on a plate reader at A_460.

Supernatants from meshes or hydrogel-meshes soaking solution mentioned before were used for inducing RAW 264.7 macrophages polarization for 48 h.
For immunofluorescence cell staining, RAW 264.7 macrophages were cultured and induced in glass bottom dishes for confocal microscopy. After being fixed with 4% PFA, cells were permeabilized with 0.1% Triton X-100, blocked in blocking buffer and incubated with APC/Cyanine7 anti-CD11b and DAPI. Fluorescence images were taken under OLYMUPS confocal microscope (Japan).
For flow cytometry, a total of 10⁶ RAW 264.7 cells per group was suspended in PBS and incubated with FITC anti-mouse CD206. Fluorescence of cells was detected under BD FACSCanto flow cytometer (USA).
For transwell migration assay, a total of 10⁵ L929 cells were seeded without serum in the upper chamber, and supernatants were in the lower chamber. After 24 h of incubation, the upper chamber was fixed in 4% PFA and stained with 0.1% crystal violet. Migrated cells were counted for analysis.
For tube formation assay, 60 μl precooled Matrigel per well was coated on a 96-well plate and solidified at 37°C for 30 min. Then, HUVECs (5× 10⁴ cells/well) were cultured and incubated with the abovementioned supernatants. After incubating at 37°C for 5 h, tube-like structures were observed under an inverted optical microscope (Leica, Germany) and the total loop numbers were counted.

Transwell assay was applied to evaluate migration ability. After treatment with metformin (0, 5, 10 and 20 mmol/L), 2 × 10⁴ MCF7 cells were suspended in serum‐free medium and seeded into the upper chamber of each well (200 μL per chamber). And then 600 μL of 10% FBS‐containing medium was added to the lower chamber. Post‐incubation at 37°C for 24 hours, the chambers were taken out and washed with PBS. The residual cells on the upper membranes were erased using a cotton swab. The remaining cells were fixed with methanol for 30 minutes at room temperature and stained with 0.2% crystal violet for 20 minutes. After washing with PBS, five fields of view were randomly selected and observed under an inverted optical microscope (Leica) to count the cells.

HUVECs were seeded into a 6-well plate and transfected with agomiR-199b-5p, antagomiR-199b-5p and miRNA scramble negative control, respectively. After 48 h post-transfection, cells with 90% confluence were pretreated with EGM-2 medium without FBS for 4 h, and then an injury line was made using a sterile 200 μl pipette and unattached cells were washed with PBS twice. Cells were allowed to migrate into the empty space for 24 h in FBS-free EGM-2. The cells were imaged during migration using an inverted optical microscope (Leica, Wetzlar, Germany). The width of the injury was measured using ImageJ software.

Cell migration of hBMSCs and HUVECs were detected by a wound healing assay. The cells were inoculated in six-well plates with the concentration of 8 × 10⁵ cells/well and treated with PBS, FNF exosomes, and NONFH exosomes. When the confluence was greater than 95–100%, the monolayer was scratched using a 10-μL sterile pipette tip and then cultured with the sterile serum-free medium supplemented with 60 μg/mL medium. The wound widths of the HUVECs and hBMSCs were observed using an inverted optical microscope (Leica, Germany) and analysed by ImageJ software.

A total of 5 × 10³ ECA109 and KYSE-70 cells were seeded in an upper chamber with serum-free medium. Concurrently, the lower chamber was filled with complete DMEM (Lonza, BE12-604F) containing 20% fetal bovine serum (FBS). After incubation for 24 h, the upper chamber was rinsed, fixed with 95% ethanol and stained with 0.1% crystal violet solution (in 20% methanol; 20 μL per well). The cells that migrated to the bottom surface of the upper chamber were depicted under an inverted optical microscope (Leica).