Live dead viability cytotoxicity test kit

To test the PDT effect, 10X concentrated QD-Ce₆ complexes maintaining QD:Ce₆ molar concentration ratio at 1:10 were prepared in a small amount of phosphate buffered saline (PBS) (pH 7.4) and kept at room temperature for 3 hours for complexes to equilibrate. Different QD-Ce₆ sols in PBS were then diluted 10 times in a serum-free cell growth medium and poured over the cells grown on Nunc Lab-Tek chambered coverglass (Thermo Fisher Scientific, USA) at a density of 25,000 cells per well. After 24 hours the cells were irradiated with 470 nm light (MAX-302 xenon light source (Asahi Spectra, Japan)) providing a 17.7 J/cm² irradiation dose in each well. After irradiation, cells were returned to the incubator. Twenty-four hours post treatment, 2 µM calcein AM and 4 µM ethidium homodimer-1 (LIVE/DEAD viability/cytotoxicity test kit by Thermo Fisher Scientific, Waltham, MA, USA) were added to each well and kept for 20 min before confocal microscopy analysis. Statistical analysis of PDT-affected cells was performed by direct counting of green (alive) and red (dead) cells in at least 5 different fields. Statistical significance was assessed using unpaired two-tailed Student’s t test.

After 72 h of cell seeding, Live/Dead viability/cytotoxicity test kit (Thermo Fisher Scientific) was used to observe the living and dead cells distribution within the spheroids. The kit includes two staining solutions, calcein AM targeting living cells and ethidium homodimer labeling dead cells. Calcein AM is hydrolyzed by esterase in living cells to become calcein, which retains in the cell cytoplasm and strongly emits green fluorescence; while ethidium homodimer only penetrates cells with disrupted plasma membrane and intercalates into DNA. Calcein AM and ethidium homodimer were mixed and diluted in PBS as instructed by the manual. After washing mASC spheroids collected from the nanosheet and the suspension culture media with PBS, 1 mL staining solution were added to each culture dish, followed by 15 min incubation at 37°C. Then, suspension cells were transferred into a glass-based dish prior to the observation under a confocal laser-scanning microscope (FV1000, Olympus Life Science). The green and red fluorescence intensity was separately analyzed by ImageJ and the ratio of living and dead cells in spheroids was compared between different groups. The corrected total cell fluorescence (CTCF) value was calculated using the formula (1) shown below: