Determination of cytotoxic concentration (CC50)

The cytotoxic concentration of porphyrins was determined by treating vero cells with 15.62 µM to 1 mM Heme, CoPPIX or SnPPIX for 1 hour at 37 °C. In light-stimulated conditions, a previous step of light exposure for 10 minutes was included. After incubation, cells were washed with PBS and cultured for 24 h in DMEM high glucose with 5% FBS. Cell viability was assessed by MTT reduction assay. Briefly, cells were incubated for1 h with 0.5 mg/mL of 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT; Life Technologies) at 37 °C. Next, the solution was removed and precipitated Formazan was diluted in isopropyl alcohol with 40 mM HCl. The solution was read on a spectrophotometer at 492 nm and 620 nm. Untreated cells were used as reference of 100% viability. The cytotoxic concentration 50 (CC₅₀) was determined by a nonlinear regression with sigmoidal profile and variable slope using Graphpad Prism (version 6.0) software.