Eclipse ti e a1

Samples of uncoated and polymer-coated recellularized arteries were washed twice with PBS and labeled with the fluorescent dye, CellTracker Green/CMFDA (Invitrogen Inc., Carlsbad, CA, USA) following the manufacturer’s instructions. Arteries were fixed with 4% paraformaldehyde in PBS for 20 min at RT and stained with DAPI (1 μg/mL) in PBS. Cells on the luminal surface were imaged by confocal microscopy (Nikon Eclipse Ti-E A1, Amsterdam, Netherlands) and analyzed using NIS-Elements software (Amsterdam, Netherlands).

The early and late apoptotic D24 cells were detected using the Annexin V-FITC kit (Beckman Coulter, USA), according to the manufacturer’s instructions with slight modifications. After treating the D24 cells with 1 or 2 mg/mL of the MeOH extract for 72 h, the cultures were stained with 1 μL of Annexin V-FITC (0.25 μg/mL) for 15 min, followed by 0.5 μL of PI (0.125 μg/mL) for 5 min in the dark. The treated and untreated cells were then observed using an inverted confocal microscope (Nikon Eclipse Ti-E A1, Japan) under 40× objective. The excitation wavelengths for Annexin V-FITC and PI used were 488 and 536 nm, respectively, while the emission wavelengths were 525 and 617 nm, respectively. Based on the principles of this technique, the normal cells would not be stained by the two dyes (Annexin V-FITC⁻/PI⁻); the early apoptotic cells would only be dyed by Annexin V-FITC (Annexin V-FITC⁺/PI⁻); the late apoptotic cells would be positive in both Annexin V-FITC and PI staining (Annexin V-FITC⁺/PI⁺).

Viability of probiotics adhered to BC was assessed by confocal laser scanning microscopy (CLSM). The samples were washed with sterile water and stained with LIVE/DEAD BacLight Bacterial Viability Kit (ThermoFisher, Waltham, MA, USA) following manufacturer’s instructions. This assay combines membrane-impermeable DNA-binding stain, i.e., propidium iodide (PI), with membrane-permeable DNA-binding counterstain, SYTO9, to stain dead and live and dead bacteria, respectively. Cell viability along the BC matrix was evaluated with a confocal microscope (Nikon Eclipse Ti-E A1, Nikon, Tokio, Japan) of the CIC-UGR equipped with 20 × objective. For acquiring SYTO9 signals (green channel), a 488 nm laser and 505–550 nm emission filter was used. For PI (red channel), a 561 nm laser and 575 nm long-pass emission filter were used. Images were analyzed with NIS Elements software (Nikon, Tokio, Japan).