Celltiter 96 aqueous mts

RPMI-1640 with L-glutamine was purchased from Biowest (Nuaillé, France). MnTnHex was synthesized and characterized at Duke University School of Medicine, according to Batinic-Haberle et al. [35 (link)]. Cisplatin, penicillin-streptomycin solution (10,000 units/mL of penicillin; 10 mg/mL of streptomycin), crystal violet, sodium bicarbonate, and O-(2,3,4,5,6-pentafluorobenzyl) hydroxylamine (PFBHA, ≥99%) were obtained from Sigma-Aldrich (Madrid, Spain). Ethanol absolute and acetic acid were purchased from Merck (Darmstadt, Germany). Sodium pyruvate was purchased from Lonza (Basel, Switzerland) and trypsin (0.25%), and fetal bovine serum (FBS) from Gibco (Eugene, OR, USA). HEPES and D-Glucose were obtained from AppliChem (Darmstadt, Germany). CellTiter 96^® Aqueous MTS (3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxylmethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) was acquired from Promega (Madison, WI, USA).
The stock solution of MnTnHex and its dilutions were prepared in Milli-Q water. Cisplatin was dissolved in saline solution (0.9% NaCl), and its aliquoted solutions were stored at −20 °C. In all cell-based assays, controls were also included, in which cells were exposed to either saline solution or Milli-Q water.

The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (MTS) reduction assay was carried out as a confirmatory assay of cell viability. This methodology is commonly used in in vitro toxicology and is based on the enzymatic reduction of a tetrazolium salt in viable cells by mitochondrial enzymes (NAD-dependent dehydrogenase) with the formation of a colored formazan product. Briefly, after incubation with the compound and removal of the complete cell culture medium, cells were washed with warm PBS, followed by the addition of 100 μL of fresh medium and 20 μL of MTS substrate prepared from the CellTiter 96^® Aqueous MTS (Promega, Madison, WI, USA) according to the manufacturer’s instructions. H1299 cells were incubated for 2 h 30 with the MTS reagent, and the absorbance was measured at 490 nm and 690 nm (reference wavelength) using a SPECTROstar OMEGA microplate reader. Absorbance values presented by vehicle-treated control cells corresponded to 100% of cell viability. Two to four independent experiments were performed, and three replicates were used for each condition in each independent experiment. The IC₅₀ was also calculated based on the concentration–response curve using GraphPad Prism^® 9.0.

CT26 cells were plated 5 × 10³ cells/well into 96-well plates. BMMCs were then plated into co-culture group wells with the ratio of 1:5 to CT26 cells. After 48 h in culture, cell proliferation was assessed using CellTiter 96 Aqueous MTS (3-[4,5-dimethyl-thiazol-2-yl] -5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazo-lium, inner salt) assay (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Briefly, reagent was added to each well and incubated at 37 °C for three hours. Cell proliferation was assessed by measuring the absorbance at 490 nm with a microplate reader (Thermo Fisher Scientific).

D-Glucose and HEPES were acquired from AppliChem (Darmstadt, Germany). Cisplatin, crystal violet (CV), extracellular matrix (ECM), penicillin–streptomycin (Pen/Strep) solution (10,000 units/mL of penicillin; 10 mg/mL of streptomycin) and sodium bicarbonate were obtained from Merck (Madrid, Spain). RPMI-1640 medium with L-glutamine was purchased from Biowest (Nuaillé, France). FBS and Trypsin (0.25%) were obtained from Gibco (Eugene, OR, USA). Sodium pyruvate was acquired from Lonza (Basel, Switzerland). CellTiter 96^® Aqueous MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-Tetrazolium) was obtained from Promega (Madison, WI, USA). Acetic acid and ethanol absolute were acquired from Merck (Darmstadt, Germany).
MnBuOE was synthesized and characterized at Duke University School of Medicine. Stock solutions and respective dilutions were formulated in Mili-Q water. Cisplatin was dissolved in saline solution (0.9% NaCl) and its aliquoted solutions were kept at −20 °C. In all cell-based assays, controls were also included, in which cells were exposed to saline solution or Milli-Q water.