Gen 5 2.0 all in one microplate reader

For adherent cultures (parental H460 cells), cells were plated in 96-well cell-culture microplates (Costar, USA) at ~2,000 cells per well and incubated overnight in CM. Then, the cells were exposed to the appropriate concentration of drug or vehicle for 72 h. Cell viability for adherent cells was evaluated by the MTT assay. The absorbance of solubilized formazan was read at 570 nm using Gen 5 2.0 All-In-One microplate reader (BioTek Instruments, Inc.).
For floating LTSs, cells growing in Poly-HEMA plates were collected in 15 mL Falcon tubes, centrifuged at 700 rpm × 3 min, and resuspended in fresh SFM. In order to plate the same number of cells, this cell suspension was split in 1 mL aliquots. Vehicle or drugs were added to each aliquot and then 150 μL cell suspension was loaded into each microwell (in a 96-well plate) and incubated for 72 h. For floating LTSs, cell viability was evaluated by the CCK-8 assay (Dojingo Laboratories).
In all cases, the highest concentration of DMSO was used in the control and this concentration was maintained below 0.001% (v/v). This DMSO concentration did not show any significant antiproliferative effect on the cell lines or LTSs in a short-term assay.

Cell viability was evaluated by the CCK-8 assay (Dojingo Laboratories, Rockville, MD, USA) that uses the water-soluble tetrazolium salt, WST-8 as follows: 15 μl of WST-8 solution was added to each microwell, incubated for 60–120 min and the absorbance was read at 450 nm using Gen 5 2.0 All-In-One microplate reader (Bio-TEK, Instruments Inc.). In parallel, microwells incubated with media alone followed by incubation with WST-8 solutions for equivalent time points were used to subtract background levels for the accurate analysis of cells viability.