Time-lapse imaging for cell proliferation assay

For assessment of cytotoxicity and phototoxicity of PC dyes, we also used commercialized red fluorescence nucleus markers (SYTO 80, SYTO 82, SYTO 84; ThermoFisher Scientific). HeLa cells were stained with each dye in DMEM ( + ) and the time-lapse observation was performed by an inverted microscope system (IX-71; Olympus) equipped with an UPlanSApo IR 20x/0.75 objective lens (Olympus), and a CMOS camera (ORCA Flash 4.0 V3 {"type":"entrez-nucleotide","attrs":{"text":"C13440","term_id":"1560993","term_text":"C13440"}}C13440; Hamamatsu photonics). The TRITC-A-Basic fluorescent filter set (FF01-542/20, FF570-Di01, FF01-620/52; Semrock Inc.) was used for all nucleus markers. The stage incubator system (Tokai Hit Co, Ltd.) was used to keep temperature at 37 °C and 5% CO₂/95% air condition. The fluorescence and bright-field time-lapse images were taken with or without excitation using an imaging software (MetaMorph; Molecular Devices) and cell proliferation rate was assessed by visual inspection from bright-field time-lapse images.