2.12. Cell Viability (MTT) and Cell Death (LDH) Assays

Cells plated in 96-well plates (10⁴ cells/well) were transfected with the aptamers, after 16–24 h, at the concentrations indicated in the figure legends using Lipofectamine^TM 2000 (Invitrogen, Boston, MA, USA) and following the instructions for siRNA transfection. The entry of the aptamers into the cells was verified with confocal microscopy (Supplementary Figure S2). After 48, 96, or 120 h (A549, H1650, and SW900, respectively), the plates were incubated for 1.5–3 h at 37 °C with MTT (Sigma, St. Louis, MO, USA) diluted 1 mg/mL in culture medium. Next, the cells were lysed with 10% SDS and 10 mM HCl overnight at 37 °C. The absorbance was read at 540 nm in an Infinite F200 spectrophotometer (TECAN).

To perform LDH assays 48, 96, or 120 h after aptamer transfection (A549, H1650, and SW900, respectively), the supernatants were collected to be mixed with the reagent of the Cytotoxicity Detection kit (LDH) (Roche, Madrid, Spain) by following the manufacturer’s protocol. The reaction was incubated for 30 min at room temperature and was stopped by adding 1 M HCl. The solubilized formazan was then measured in an Infinite F200 spectrophotometer at 490 nm. To calculate the percentage of cytotoxicity, two controls were included: the supernatants of the untreated cells (LDH_low) and of the lysate cells (LDH_high) with 0.2% Triton X-100. Finally, the results are expressed as cytotoxicity (%) = ((LDH_treated − LDH_low)/(LDH_high − LDH_low) × 100 for each experimental point respective to the control.