Living/Dead viability assay by calcein AM and propidium iodide

Cell viability was assessed by calcein-AM (living) and propidium iodide (PI) (dead) double staining. The double staining solution was prepared with 2 μM calcein-AM and 4.5 μM PI in PBS. Seeding of ESI017 was performed as described above in the section Cell seeding on micropatterned surface. The cells were cultured in mTeSR1 medium with ROCKi at 37°C for 24 hours. Next, the staining solution was added into each well and incubated at 37°C for 15 minutes. After washing, the cell colonies on micropatterned slides were imaged using multi z-sections at 10X resolution NA 0.40 on an Olympus FV31S-SW laser scanning confocal microscope. Cell counting was performed by image analysis with FIJI [19] and ilastik [20], following the steps: making max intensity projection and splitting channels in FIJI, creating binary masks for the live cell and dead cell channels in ilastik, performing watershed and analyzing particles on the cell masks in FIJI. The viability was calculated as living cell count over total cell count (= living cell count + dead cell count). ESI017 cells growing on an LN521 coated glass surface were used as a positive control.