Live dead cytotoxicity assay for mammalian cells

Cytotoxicity test was performed, as has been mentioned elsewhere by Thi et al. [31 ], towards HDF via LIVE/DEAD cytotoxicity assay for mammalian cells (Thermo Fisher Scientific, Waltham, MA, USA). The hydrogels (n = 3) were fabricated in a 48-well polystyrene culture plate by using sterile gelatin and genipin solution. Immediately after polymerisation, 5 × 10⁴ HDF passage three were seeded on the top of hydrogel prior to the incubation for 24 h. Cell toxicity was examined by using a fluorescence microscope (Nikon A1R-A1, Japan) at 100× magnification after treatment with 500 µL of a mixture of 2 mM acetomethoxy derivate of calcein (calcein-AM) and 4mM ethidium homodimer-1 (EthD-1) at 37 °C for 30 min.

Fmoc-Phe-Phe peptide powder with a purity greater than 95% was purchased from Bachem (Bubendorf, Switzerland). The porphyrins H_2-T(MePy)P(I₄), Zn-T(MePy)P(I₄), and Zn-T(MePy)P(Cl₄) were synthesized as previously described [43 (link)]. The NIH3T3 cell line was cultured at 37 °C, 5% CO₂ in DMEM (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) medium supplemented with 10% fetal bovine serum (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and 50 μg/mL gentamycin (Applichem, Darmstadt, Germany). Thiazolyl blue tetrazolium bromide (MTT), isopropanol, dimethylsulfoxide (DMSO), ethanol, hexamethyldisilane (HMDS), and phosphate-buffered saline (PBS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Live/Dead cytotoxicity assay for mammalian cells was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Tissue culture (TC) inserts with a 0.4 μm pore size were purchased from Sarstedt (Nümbrecht, Germany).