Resazurin cell viability assay kit

Resazurin Cell Viability Assay kit (Abcam, Waltham, MA, USA, ab129732) was used to determine if the concentrations of IMQ, RSQ, and GDQ used in experiments were cytotoxic to the cancer cell lines under experimental conditions. To begin, cancer cells were plated in complete cell media (DMEM, 10% HI-FBS) in 96-well plates, with two rows per plate of each density/well tested: 2000 cells/well, 25,000 cells/well, 50,000 cells/well, and 100,000 cells/well. Cells were treated with 20 μL of 1 mM solutions of IMQ, RSQ, and GDQ, to give a final concentration per well of 100 μM for each compound. Plates were incubated at 37 °C for 1 h. After incubation, 10 μL of resazurin stain was added to wells in alternating rows on the plate, such that for each density and compound tested in triplicate there was a corresponding blank without stain added. Plates were incubated further with absorbance measured at 570 nm and 600 nm at 1, 2, 3, 4, and 24 h intervals. The absorbance measurements at 600 nm, which correlated to the absorbance of resazurin, were subtracted from those taken at 570 nm, correlating to the resorufin absorbance. After this, values were normalized to cell viability of the negative control. Experiments were performed in triplicate and the 3 h time-point for 5 × 10⁵ cells is shown (Figure S4).

Cell viability was analyzed by using the Resazurin Cell Viability Assay Kit (Abcam; cat # Ab129712, Milano, Italy), following the manufacturer’s protocol. Briefly, cells were seeded in 96-well plates on top of the extracellular matrix gel prepared as described above. After 24 h, cells were incubated with GEM or C18 for 96 h. At the end of the treatment, 15 μL of resazurin was added directly in the medium of each 96-well for 3 h at 37 °C and the fluorescent signals were detected at Ex/Em 535/590 nm by using a Varian Cary Eclipse Fluorescence Spectrophotometer (Agilent Technologies, Santa Clara, CA, USA). For each treatment, the obtained data were normalized with the respective control group.