Axio observer zi

Microfluidic devices were imaged with fluorescence microscopy (Zeiss Axio Observer ZI with Zen Pro software suite) equipped with Apotome.2 (Zeiss) at 10× and 20× magnification. Phase contrast images were uploaded into NIH ImageJ software (NIH ImageJ software) and processed, while fluorescent images were processed in both ImageJ and Zen Pro Imaging software. Besides mono‐cultured ECs which were captured in the vascular region, all IF images were captured within the stroma layer of the device.

MSCs encapsulated or seeded on hydrogels were evaluated for viability using an ethidium homodimer-1/calcein acetoxymethyl (AM) LIVE/DEAD® Viability/Cytotoxicity Kit (ThermoFisher, Waltham, MA, USA). For the long-term viability study assessing hydrogel biocompatibility, seeded cells were stained for viability on days 1, 4, 7, 10, 14, and 22. To assess cell survival following hydrogel actuation, viability stains were performed at several timepoints after electrical stimulation: 24 hrs, 48 hrs, and 72 hrs. After each timepoint, fresh dye solution containing calcein AM (2 μM) and ethidium homodimer-1 (4 μM) was prepared. Media was aspirated out, and 1 mL of the dye solution was added to each well (n = 4) and incubated for 10 minutes in a humidified incubator with 5% CO₂ at 37°C before imaging with an epifluorescent microscope (Axio Observer ZI, Zeiss). Cells were observed at 10x magnification with 2 fluorescent channels that labeled cells live (green, ex/em; ∼450/475 nm) and dead (red, ex/em; ∼600/635 nm). Hydrogels were imaged in triplicate.

Immunofluorescence microscopy was performed to detect resistant proteins like the p-glycoprotein (P-gp) qualitatively. Briefly, cells were grown on a coverslip, fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS, blocked for non-specific binding with 10% mouse serum in PBS, and exposed to an unlabeled primary anti-P-gp antibody. The slides were washed and incubated for 1 h at room temperature with a secondary fluorescence-labeled antibody that binds to the primary antibody. The cell nuclei were counterstained with 1 μg/ml 4′,6-diamidino-2-phenylindole (DAPI). The coverslip was inverted onto a glass slide, mounted, and observed under a Carl Zeiss Axio Observer ZI fluorescence microscope.

An EdU (RiboBio, China, C10310-1) assay was used to determine the cell proliferation rate. Cells were seeded in 96-well plates (1×10³ cells/well) and cultured with DMEM (10% FBS) for 48 h. Then, 100 µl of EdU solution (50 µM) was added to each well and incubated for 2 h at 37°C. Then, the cells were fixed with 4% formaldehyde for 30 min and permeabilized with 0.5% Triton X-100 for 10 min at room temperature. Next the cells were washed with PBS for 5 min and 1X ApolloR reaction cocktail was added to each well (100 µl/well). Thirty minutes later, the cells were incubated with 100 µl Hoechst 33342 for 30 min. Finally, the cells were observed with an inverted microscope (Zeiss, Axio Observer. ZI, USA).

Cells were plated at a density of 1×10⁴ cells/wells in 24-well plates. After cells were attached overnight, AsA at indicated concentrations was added to each well for 12 h. Then, the cells were incubated with 5-ethynyl-20-deoxyuridine (EdU, Ribobio) for 2 h, and processed according to the manufacturer’s instruction. After three washes with PBS, the cells were treated with 300 μL of 1× Apollo reaction cocktail for 30 min. Then, the DNA contents of the cells in each well were stained with 200 μL of Hoechst 33,342 (Sigma-Aldrich, Germany) (5 μg·mL⁻¹) for 30 min and visualized under a fluorescence microscope at 20× magnification (Axio Observer ZI, Carl Zeiss AG, Oberkochen, Germany).