Celltiter 96 cell proliferation assay

Cell viability assays were performed using the tissue culture plates described above to provide a quantitative measure of cell viability of toxin-treated cells that had been pre-treated with sialidase-containing culture supernatants. To perform these assays a Celltiter 96 cell Proliferation Assay (Promega, WI, USA) was used as per manufacturer’s instructions with slight modifications. The tetrazolium compound 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethopheny)-2-(4-sulfophenyl)-2H-tetrazolium inner salt, or MTS (Sigma) was prepared at 2 mg/mL in Dulbecco’s Phosphate Buffered Saline (2.7 mM KCl, 1.5 mM KH₂PO₄, 136.9 mM NaCl, 8.9 mM Na₂HPO₄.7H₂O) (DPBS) and the electron coupling reagent (phenazine methosulfate) PMS (Sigma) was prepared at 0.92 mg/mL in DPBS. Before use, 100 μL of PMS was mixed with 2 mL of MTS and 20 μL of this solution was added to each well containing 100 μL of tissue culture medium. Plates were then read after a 1 h incubation at 37 °C in a 5% CO₂ incubator, using a Tecan infinite M200 plate reader. This assay measures cell viability or activity since the absorbance at 490 nm is directly proportional to the number of living cells. The data is presented as the average of between three to five independent experiments, each performed in duplicate, and is plotted as a percentage of cell death versus toxin titre.

Cell cytotoxicity was evaluated using CellTiter 96 cell proliferation assay (Promega, Madison, WI, USA). Cells (5 × 10⁴ /well) were treated with free Dox, AS1411, control DNA-Dox adduct, and AS1411-Dox adduct (respectively) in FBS-free medium. All treatments were suspended in PBS. After incubation for 1 h (37°C, 5% CO₂), medium was removed, and fresh medium (10% FBS, 100 IU/mL penicillin-streptomycin, 200 μL) was added for further cell growth (48 h). Then medium was removed again, and CellTiter reagent (20 μL) diluted in fresh medium (100 μL) was added to each well and incubated for 1–2 h. The absorbance (490 nm) was recorded using a microplate reader (Tecan Safire microplate reader, AG, Switzerland). Cell viability was determined according to the manufacturer’s description.

Growth of cells was assayed using the Promega CellTiter 96 Cell Proliferation Assay. Cells were resuspended to a final concentration of 1.0 x 10⁵/mL in RPMI. 50 μl of the cell suspension (5,000 cells) was added to each well of the 96-well plate containing 50 μl of media with corresponding treatment resulting in a total volume of 100 μl. The micro plates were incubated at 37°C for 24–72 hours in a humidified, 5% CO2 atmosphere. Per manufacturer’s instructions, following incubation, 20 μl of MTS / PMS solution was added to each well and incubated for 4 hours. Absorbances were read at 490 nm using the Synergy H1m multimode micro plate reader.