96 well polystyrene microplates

The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)colorimetric test was used to determine the antiproliferative effect of 1C and N-acetylcysteine. HCT116 cells (5 × 10³/well) were seeded in 96-well polystyrene microplates (SARSTEDT, Nümbrecht, Germany). Twenty-four hours after seeding, 1C final concentration (10 µM) and NAC (final concentration 0.3, 0.5, 1, 1.5, 2, and 2.5 mM) or their combinations were added. After 72 h, cells were incubated with 10 μL of MTT (5 mg/mL, Sigma-Aldrich Chemie, Steinheim, Germany) at 37 °C. After an additional 4 h, insoluble formazan produced by metabolic reactions were dissolved by 100 μL of a 10% sodium dodecyl sulphate. Cell proliferation was evaluated by measuring the absorbance at wavelength 570 nm using the automated Cytation 3 Cell Imaging Multi-Mode Reader (Biotek, Winooski, VT, USA). Absorbance of control wells was taken as 1.0 = 100%, and the results were expressed as a fold/percentage of untreated control.

Cell proliferation activity was directly monitored by quantifying BrdU incorporated into the genomic DNA during cell growth. DNA synthesis was assessed using a colorimetric cell proliferation ELISA assay (Roche Diagnostics GmbH, Mannheim, Germany), following the vendor’s protocol. Briefly, the tested cell lines, at the same density as in the MTT viability assay, were plated in 96-well polystyrene microplates (Sarstedt AG & Co, Nümbrecht, Germany) with 80 µL of medium. Twenty-four hours after cell seeding, different concentrations (1, 3, 5, 7 and 10 μmol/L) of the compound MB-591 were added. After 48 h of treatment, cells were incubated with BrdU labeling solution (10 µM final concentration) for an additional 24 h at 37 °C, followed by fixation and incubation with anti-BrdU peroxidase conjugate for an additional 1.5 h at room temperature (RT). Finally, after the TMB substrate reaction, the stop solution (25 µL 1 mol/L H₂SO₄) was added. The change in absorbance was analyzed at 450 nm (for yellow) and 690 nm (for blue) using the automated CytationTM 3 Cell Imaging Multi-Mode Reader (Biotek, Winooski, VT, USA). Three independent experiments were performed.

The MTS colorimetric test [21 ] was used to determine the antiproliferative effect of CBE and N-acetylcysteine. MCF-7 cells (1 × 10⁴/well) were seeded in 96-well polystyrene microplates (SARSTEDT, Nümbrecht, Germany). Twenty-four hours after seeding CBE final concentrations 350 and 450 μg/mL and NAC (final concentration 1.5 mM) or Z-VAD-FM (50 µM as pre-treatment; Enzo Life Sciences, Inc., Farmingdale, NY, USA) or their combinations were added. After 72 h cells were incubated with 10 μL of MTS (5 mg/mL) at 37 °C. After an additional 2 h, cell proliferation was evaluated by measuring the absorbance at wavelength 490 nm using the automated Cytation™ 3 Cell Imaging Multi-Mode Reader (Biotek, Winooski, VT, USA). Absorbance of control wells was taken as 1.0 = 100%, and the results were expressed as a fold/percentage of untreated control. All experiments were performed in triplicate.

The cytotoxic effects of orotic acid, sodium salt of orotic acid, and the Nd(III) complex were determined using a colorimetric microculture assay with the MTT end-point [63 (link)]. Briefly, 3 × 10³ (A-549, MCF-7, MDA-MB-231), 5 × 10³ (HeLa), or 1 × 10⁴ (Jurkat and CEM) cells were plated per well in 96-well polystyrene microplates (Sarstedt, Germany) in the culture medium containing the tested chemicals at final concentrations of 10⁻⁴–10⁻⁹ mol·L⁻¹. After 72 h of incubation, 10 μL of MTT (5 mg × mL⁻¹) (Sigma, Darmstadt, Germany) were added to each well. After an additional 4 h, during which insoluble formazan was produced, 100 μL of 10% sodium dodecyl sulphate were added to each well, and another 12 h were allowed for the dissolution of the formazan. Absorbance was measured at 540 nm using an automated MRX microplate reader (Dynatech Laboratories, Chichester, UK). The blank-corrected absorbance of the control wells was taken as 100%, and the results were expressed as a percentage of the control. All experiments were performed in triplicate.