Celltiter 96 aqueous one solution proliferation assay kit mts

Human cancer cell lines including lung (H1299), cervical (HeLa, SiHa and CaSki), colon (Caco-2), and liver (Hep G2), were used to conduct cell viability assays. Cancer cell lines were cultured in RMPI-1640 and EMEM medium, both supplemented with 10 % of foetal bovine serum (FBS, Sigma-Aldrich) and 1 % antimycotic antibiotic (Sigma-Aldrich). Cells were incubated at 37 °C with 5 % CO₂. Cell viability assay was evaluated by the cytotoxic colorimetric reaction CellTiter 96 Aqueous One Solution Proliferation Assay Kit (MTS) (Promega). The MTS reaction is carried out by enzymes in living cells that convert tetrazolium salt into a soluble formazan product that is coloured. For each cancer cell line, a total of 5×10⁴ cells per well were added to culture plates and were incubated during 24 h at 37 °C with 5 % CO₂. After the incubation period, 50 µg of each crude extract were added to culture plates in triplicate, and 1 % DMSO (vehicle) was used as negative control (C-) and 50 µg of etoposide was used as positive control (C+). Plate was incubated for 24 h, followed by an addition of 20 µl MTS to each treatment, and absorbance was measured at 490 nm, using a microplate reader EPOCH (Biotek, Winooski, United States), in order to determine the number of viable cells. All results were normalized to C-.

Cytotoxicity was evaluated using a colorimetric reaction named CellTiter 96 Aqueous One Solution Proliferation Assay Kit MTS (Promega). In the MTS reaction, a tetrazolium salt is converted into a soluble formazan product (colored) by enzymes in live cells. The amount of formazan is directly proportional to the number of viable cells. A total of 5 × 10⁴ cells per well were added to culture plate. Cells were incubated 24 h at 37 °C with 5% CO₂. A total of 27 and 28 μM of Cal14.1a and Cal14.1b were added, respectively. As the positive control (C+) staurosporine was used at 5 μM (dissolved in 1% DMSO). For the negative control (C-), cells were treated with 1% DMSO vehicle. After 24 h incubation, 20 μL of MTS were added to each treatment and the number of viable cells were determined by measuring absorbance at 490 nm on microplate reader EPOCH (BioTek, Winooski, United States).