seeded onto the hydrogels and incubated for 4 days. The medium was changed once every 2 days. After 4 days of incubation, the medium was removed and the cells were washed with ice-cold 1 × PBS. Ten milligrams of fluo- rescein diacetate (FDA) (Sigma Aldrich) was weighedand dissolved in 10 mL of acetone, which served as the stock solution. A working solution was prepared freshly by mixing 30 µL of the stock solution with 10 mL of PBS. One milliliter of this working solution was added onto the films in each well and incubated for 15 min at 37°C in the dark [51]. Cells were observed under a fluorescent microscope with 4× and 10× objectives.
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