Methyl thiazolyl tetrazolium (mtt)

Cytotoxicity of Zn-Pheide, both in the dark and after irradiation, was determined by MTT assay. For cytotoxicity studies, 3 × 10⁴ cells were seeded onto 96-well plates. After an overnight growth, the cells were incubated in the dark for 3 h with Zn-Pheide at various concentrations. Control cells were incubated with the appropriate concentration of DMSO (below 0.5%) without the PS. For PDT, the cells were rinsed with PBS, covered with HBSS, and illuminated with 2 J/cm² delivered by a LED illuminator equipped with a 600 nm cut-off filter. After that, they were covered with complete culture medium and incubated for another 24 h. Then, 0.5 mg/mL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), (Carl Roth, Karlsruhe, Germany) was added to each well, and the cells were incubated for another 3 h. The precipitated formazan crystals were dissolved in an ethanol/DMSO mixture (v/v, 1:1) and the absorbance of the solutions at 570 nm was measured using a SpectraMax i3 plate reader (Molecular Devices, San Jose, CA, USA). The signal from the treated cells was compared to that of the vehicle-only control cells (100%) to calculate the percentage viability. All experiments were performed in triplicates. Dose–response curves were fitted and IC50 values determined in Origin 2021 [51 ].

Cells were seeded onto 96-well plates (8000 cells/100 μl per well) and were allowed to attach overnight. Then cells were treated with 50,100 and 150 μM TMZ for 3 days. Cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl- tetrazolium-bromide (MTT, 5 mg/ml in phosphate-buffered saline; Roth, Karlsruhe, Germany) for 2–3 h. Afterwards, the formazan crystals were lysed with 100 μl acidic isopropanol and the absorbance of the obtained solutions was determined at 570 nm using TECAN F50 (Crailsheim, Germany) software.

Allicin was diluted in RPMI 1640 medium to the concentrations as indicated. 50 µL of allicin was mixed with 50 µL cell suspension (in RPMI 1640) in wells of 96-well plates (Sarstedt, Nuembrecht, Germany). All treatments were performed in triplicate. The final cell number was 1 × 10⁵ cells per well. Cells were incubated for three days at 37 °C and 5% CO₂. 50 µL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Carl Roth GmbH, Karlsruhe, Germany, 1% w/v in PBS) was added and incubated for three hours at 37 °C and 5% CO₂. Cells were collected by centrifugation (300×g), washed with PBS and the supernatant was removed. 100 µL 2-propanol was added and plates were incubated for 15 min under shaking to solve the formazan salt. The measurement was performed at 570 nm in a microplate reader (Sunrise plate reader, Tecan, Crailsheim, Germany.)

The cell metabolic activity was measured by an MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) assay. PTEC and HUVEC were seeded at 3 × 10⁵ cells/mL; after 24 h, VP1u at concentrations of 50, 100 and 200 µg/mL was added to fresh media and left to incubate at 37 °C with 5% CO₂ overnight. After the incubation, the growth media were removed, and the cells were rinsed once with PBS. In total, 0.2 mg/mL MTT (Sigma-Aldrich, Darmstadt, Germany) was dissolved in the media and added to cells, and was then incubated for 1 h at 37 °C. Then, the MTT solution was discarded, and water-insoluble formazan was dissolved in dimethyl sulfoxide (DMSO) (Carl Roth, GmbH, Germany) with gentle shaking at RT for 10 min. The results were quantified using a spectrophotometer Varioskan Flash (Thermo Fisher Scientific, Waltham, MA, USA), reading absorbance at 570 nm.

Cell viability was measured using MTT assay. In all, 5000 cells per well were seeded on a 96-well plate one day prior to the experiment. The following day, the cells were treated with different compounds as described above. After that, 0.5 mg/ml MTT (Roth, #4022) was added to the cells, and cells were incubated at 37 °C for 1 h. Then the plates were centrifuged at 600 rcf for 5 min, and formazan crystals were dissolved in DMSO for 20 min in dark. The absorbance was measured at test (570 nm) and reference (650 nm) wavelengths using a microplate reader (BioTek, Synergy Mx). The mean of the absorbance of untreated control samples was set as 100%.