Labsystems multiscan rc

The number of proliferating cells was determined by measuring the amount of 5-bromo-2′-doexyuridine (BrdU) incorporated into newly synthetized DNA using the BrdU Cell Proliferation Assay (Merck Millipore, USA). Forty eight hours after transfection with siRNA, the cells were seeded in 96-well plates (5 replicates) and BrdU was added to each well. Next, the cells were incubated at 37 °C for 17 h in a 5% CO₂ incubator and then treated following the manufacturer’s instructions. Absorbance was measured at the test wavelength of 450 nm and the reference wavelength of 595 nm in the Labsystems Multiscan RC microplate reader (Thermo, Finland).

A total of 2.5 × 10⁴/mL growing cells were seeded into each well of the 96-well plates and compounds were added at 0.5–50 µmol/L. The solvent, DMSO at a concentration of 0.28%, was also applied as a control (Sigma-Aldrich, St Louis, MO, USA). Two duplicates were created for each concentration with a total volume of 100 µL per well. Then, 10 μL of MTT solution (5 mg/mL) was added to each well. The plates were incubated at 37 °C for 4 h followed by the addition of 100 μL solubilization buffer (10% SDS in 0.01 M HCl). Cell viability was quantified spectrophotometrically using a Labsystems Multiscan RC (Thermo, Champaign, IL, USA). Each experiment was repeated three times, IC₅₀ values were calculated using the CampuSyn software (ComboSyn, Paramus, NJ, USA), and the standard deviation was calculated using the Microsoft Excel software (Microsoft, Redmond, WA, USA) [9 (link),68 (link)].

Into each well of the 96-well plates, 2.5 × 10⁴/mL growing cells were seeded and compounds were added at the concentration range of 0.5–50 µmol/l. The solvent, DMSO at a concentration of 0.28%, was also applied as a control (Sigma-Aldrich, St. Louis, MO, USA). Two biological duplicates with three technical repeats were created for each concentration with a total volume of 200 µL per well. Then, 20 μL of MTT solution (5 mg/mL) was added to each well. The plates were incubated at 37 °C for 4 h followed by the addition of 100 μL solubilization buffer (10% SDS in 0.01 M HCl). Cell viability was quantified spectrophotometrically using a Labsystems Multiscan RC (Thermo, Champaign, IL, USA). Each experiment was repeated two times, IC50 values were calculated using CampuSyn software version 2022 (ComboSyn Inc., Parammus, NJ, USA), and the standard deviation was calculated using Microsoft Excel software (Microsoft, Redmond, WA, USA).