4.2.3. Cell Viability of 3D Cell Culture

Cell viability in the alginate beads was tested using calcein/ethidium homodimer staining. Staining solution was prepared by mixing culture media with 2 μM ethidium homodimer (46043, Sigma) and 2 μM calcein-AM (17783, Sigma). 200 μL/well of the staining solution was added into a 96-well plate containing beads (one bead per culture condition per well) and then incubated for 30 min. Subsequently, the beads were gently squeezed between cover slips and three photos were randomly captured with a fluorescence microscope (Olympus IX51, Tokyo, Japan) at the wavelength of 515 nm (calcein: living cells) and 620 nm (ethidium: dead cells). The cell numbers in each image were counted by ImageJ ver.1.51j8 and averaged. Cell viability is shown as the number of living cells per total cells.
