Cell proliferation reagent wst 1 assay

Compounds were evaluated for their antiproliferative activity using NHDF (normal human dermal fibroblasts, ATCC, Manassas, VA, USA). The cultured cells were kept at 37 °C and 5% CO₂. The cells were seeded (1 × 10⁴ cells/well/100 μL DMEM supplemented with 10% FCS and streptomycin/penicillin) into 96-well plates (Corning Inc., Corning, NY, USA). Cells were counted using a hemocytometer (Burker chamber) and phase contrast Olympus IX50 microscope equipped with Sony SSC-DC58 AP camera and Olympus DP10 digital camera. The cell viability of the compounds was determined using the Cell Proliferation Reagent WST-1 assay (Roche Molecular Biochemicals, Mannheim, Germany). The examined cells were exposed to the tested compounds (1 mg/mL DMSO stock) for 72 h at various concentrations (0.1–100 μg/mL). The control was included in order to eliminate the DMSO effect at the concentration used. Cell cultures were incubated with WST-1 (10 μL) for 1 h. The absorbance of the samples was measured against a background control at 450 nm using a microplate reader with a reference wavelength at 600 nm. The obtained results are expressed as means of at least two independent experiments performed in triplicate. The values of IC₅₀ (compound concentration required to cause 50% inhibition) were calculated from the dose–response relationship with respect to control.

Cells (MDA-MB-231 or MDA-MB-231BR, 5000/well) were seeded in quintuplicates in 96-well-plates, grown for 24h and treated with Ipatasertib (1, 5, 10 or 20µM) or 0.1% DMSO as control by replacing the cell culture medium for 24, 48 and 72h. MDA-MB-231BR AKT1_KO or Ctrl cells were seeded in quintuplicates as described above in cell culture serum-reduced medium and grown for 24, 48 and 72h. Cell viability was determined by Cell Proliferation Reagent WST-1 assay (Roche, Mannheim, Germany) using the manufacturer’s protocol and Microplate Absorbance Reader (Tecan Trading AG, Schweiz). Cell viability values of each cell line after 2 hours were used for normalization. For statistical analyses, each experiment was performed three-times (n=3) and results were given as mean ± S.D.

Cell viability was assessed using the Cell Proliferation Reagent WST-1 assay (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer’s instructions. DCs resting or activated with R848 were treated with oxyresveratrol alone or encapsulated in PLGA nanoparticles, or with corresponding amounts of bare PLGA particles for 24 h. After treatment, cell supernatant was removed and 50 µL of pre-warmed fresh complete medium were added to the cells and to 3 empty wells (Blank). A 2× WST solution was freshly prepared by dilution of the 10× WST reagent in the complete medium and a volume of 50 μL was dispensed in the wells and blank. The plate was incubated for 60 min. The absorbance (OD) of the samples was measured using a Victor3 multilabel reader (PerkinElmer, Shelton, CT, USA) at 450 nm.

Cell viability was assessed using the Cell Proliferation Reagent WST-1 assay (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer’s instructions. Monocytes resting or activated with β-glucan were treated with oxyresveratrol alone or encapsulated in PLGA nanoparticles, or with corresponding amounts of unloaded PLGA particles for 18 h. After treatment, the cell supernatant was removed and 50 µL of pre-warmed fresh complete medium were added to cells and to three empty wells (blank). A 2× WST solution was freshly prepared by dilution of the 10× WST reagent in the complete medium and a volume of 50 μL was dispensed in the wells and blank. The plate was incubated for 60 min. The absorbance (OD) of the samples was measured using a Victor3 multilabel reader (PerkinElmer, Shelton, CT, USA) at 450 nm.

Antiproliferative effect of compounds was determined using the Cell Proliferation Reagent WST-1 assay (Roche Diagnostics, Mannheim, Germany). After exposure to tested compounds (at concentrations between 0 and 100 μg/ml) for 72 h, cells were incubated with WST-1 (10 μl) for 1 h, and the absorbance of the samples against a background control was read at 450 nm using with a reference wavelength λ = 600 nm a microplate reader. Results are expressed as means of at least two independent experiments performed in triplicate.