Cytotoxicity assay on Vero E6 cells

The cytotoxicity of porphyrin and por-CDs on Vero E6 cells was evaluated using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay, which quantifies the viable cells after treatment of the tested compounds. Vero E6 cells were seeded in 96-well plates (100 μL/well) at a density of 2 × 10⁴ cells/well and incubated at 37 °C with 5% CO₂. After incubation for 24 h, the culture medium was discarded, followed by the addition of the diluted compounds to cell monolayers (100 μL/well). For 0 μg/mL porphyrin treatment (vehicle control), cells were treated with 1% DMSO-contained medium. The assay was performed with three replicates (n = 3). The plates were incubated for 72 h at 37 °C with 5% CO₂. At the end of the treatment, the supernatant was discarded, then the cells were washed with phosphate buffer saline (PBS). MTT (0.5 mg/mL) was added 100 μL/well and incubated for 3 h, at 37 °C with 5% CO₂, in dark condition. Finally, 100% DMSO was added (100 μL/well) to dissolve the formazan crystals formed. After 10-min incubation at room temperature, the absorbance was read at 570 nm using Varioskan™ LUX Multimode Microplate Spectrophotometer (ThermoScientific, USA). The absorbance value of each sample was normalized by blank before the cell viability was calculated as follows.

The tested compounds at certain concentrations which resulted in cell viability of ≥ 70% were considered non-toxic [33]. The 50% cytotoxicity concentration (CC₅₀) values of porphyrin and por-CDs were obtained by linear regression calculation.