Prestoblue pb cell viability reagent

Lung cancer cells seeded in 96-well plates (2500 cells/well) were dosed 24 h later with different inhibitors for 72 h. Cell viability was determined by PrestoBlue (PB) Cell Viability Reagent (ThermoFisher Scientific) by following the manufacturer’s instructions [14 (link), 31 ]. The PB reagent was added into media directly (1:10 dilution) and incubated for 30 min-2 h and then the fluorescence was read (excitation 570 nm; emission 600 nm) at recommended time of incubation. The efficacy of drugs on cell growth was normalized to untreated control. Each data point was generated in triplicate and each experiment was done three times (n = 3). Best-fit curve was generated in GraphPad Prism [(log (inhibitor) vs response (−variable slope four parameters)]. Error bars are mean ± SD. The combination index (CI) was calculated by ComboSyn software (ComboSyn Inc., http://www.combosyn.com/).
Clonogenic assay was done as we described previously [14 (link), 31 –33 (link)]. In brief, cells seeded in 6-well plates (3000 cells/well) were dosed 24 h later and continually treated with rapamycin for 7 days (refresh drugs every 3 days), the resulting colonies were stained with crystal violet (0.5% dissolved in 25% methanol).

For 2D cultures, A549 cells were seeded in a 384-well at 600 to 900 cells/well and grown in the full DMEM medium + 1 μg/ml puromycin for 3 days before viability test using the PrestoBlue (PB) cell viability reagent (ThermoFisher) per vendor’s protocol. Briefly, 2X PB + 4 μg/ml Hoechst dye in SM (20 μl) was added to each well containing cells in 20 μl SM medium. The plate was incubated at 37 °C/5% CO₂ for 30 min before reading using excitation/emission wavelengths of 560/590 nm for PB and 360/460 nm for Hoechst.
For 3D cultures in Matrigel, viability testing was performed similarly as that for 2D cultures except for the addition of 40 μl of the test reagent to spheroids in 30 μl SM medium + 10 μl Matrigel to make final 1X test reagent and incubation at 37 °C/5% CO₂ for 1 h with gentle rocking to facilitate exchange.