Imdm medium

Ruthenium complexes 1–3 were synthesized as previously described (1) [21 (link)], (2) [22 (link)], (3) [5 (link)]. IMDM medium and fetal bovine serum (FBS) were obtained from Biowest (Cytogen, Zgierz, Poland). Ruthenium chloride (RuCl₃), 2′,7′-dichlorofluorescein (H₂DCFDA), Hank’s balanced salt solution (HBSS), dimethyl sulfoxide (DMSO), and hydrogen peroxide (H₂O₂) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Succinimide was purchased from Fluka. All other chemicals were of the highest commercial grade available. A stock solution of complexes and succinimide (10 mM) was dissolved in DMSO.

Human colorectal carcinoma HCT116 cells (#91091005, ECACC) were grown in an IMDM medium (Biowest) supplemented with 2 mM glutamine, 100 IU/mL penicillin, 100 μg/mL streptomycin and 10% FBS (Gibco). The cell lines were grown at 37 °C in a humidified incubator containing 5% CO₂. The cells were verified to be mycoplasma-free and passaged < 3 months. The inhibition of cell proliferation was measured by colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay [45 (link)]. GI₅₀ concentrations were determined using the CompuSyn software version 1.0 (ComboSyn, Inc., Paramus, NJ USA) (free download via www.combosyn.com, accessed on 30 May 2022), and statistical analyses were performed using the GraphPad PRISM software (version 6.01 for Windows, GraphPad Software, San Diego, California USA GraphPad Prism) using unpaired t-test. Data were considered to be statistically significant if p < 0.05 (*), p < 0.01 (**), p < 0.001(***) and p < 0.0001 (****).

All cell lines mentioned below were cultured at 37 °C and 5% CO₂ in a humidified Forma Steri-cycle i160 CO₂ incubator (Thermo Fischer Scientific) and were grown in the described media unless otherwise specified. HFF (ATCC® CRL-2429™, male), passage 3–6, were grown in IMDM medium (Biowest) supplemented with 10% Gibco™ Fetal Bovine Serum, Qualified, US Origin, Standard (Sterile-Filtered) (Gibco). RKO cells (ATCC® CRL-2577™, female), passage 3–5, were grown in DMEM supplemented with 2 mM glutamine (Lonza) and 10% FBS. HT29 (ATCC® HTB-38™, female), passage 3–5 were grown in RPMI (Lonza) supplemented with 10% FBS. ESCs, wild-type H9 (WiCell WA-09, female)^{61 (link)}, passage 35–40 and HS980, passage 9–25 were grown on LN-521-coated dishes in NutriStem medium (Biological Industries).