Gelcount

To assess difference in proliferation rate, cells were seeded in two black 96-well plates. Twenty-four hours after seeding, compounds were added to plates. The first plate was read as a reference after 24 h using CellTiter-Glo (Promega), according to the manufacturer’s protocol, on Synergy 2 Microplate Reader (BioTek). The second plate was read in the same way after 6 days of incubation. For clonogenic assay, cells were treated with compounds for 10 days after which they were seeded into 6-well plates, 1000 live cells per well. After 2 weeks of culture, cells were fixed with 4% paraformaldehyde and stained with crystal violet solution (Sigma). Colonies were counted with Oxford Optronics Gelcount.

Cells were plated at 500 cells/well in 96-well plate (Sarstedt), cultured for 24 h before adding drugs and further incubated for 10 days. After 10 days, the spheres were stained using Thiazolyl Blue Tetrazolium Bromide (Sigma-Aldrich) 4 h prior to image acquisition and counting using an automated colony counter (GelCount, Oxford Optronics). Spheres > 30 µM in diameter were included in the final analysis, and results are reported relative to negative control.

Cells were seeded into 6-well plates and allowed to adhere overnight. Treated cells were seeded at a density of 1000 cells per well, and mock-treated cells were seeded at a density of 500 cells per well. Cells were treated with IC₅₀ doses of cisplatin or 5-FU for 24 h, or treated with 2 Gy X-ray radiation. Post-treatment cells were incubated for 8–14 day to allow colonies to form. Colonies were fixed and stained with crystal violet (70% methanol, 30% H₂O, 0.1% w/v crystal violet) for 1 h at room temperature, followed by destaining in H₂O. Air-dried plates were imaged and colonies were counted using a GelCount instrument (Oxford Optronics, UK). The plating efficiencies and surviving fractions were calculated as previously described [26 (link)]. Colonies that could not be accurately defined and counted using the GelCount were analysed by solubilising the crystal violet stain with a 1% SDS solution overnight at 37°C. The absorbance of the resultant crystal violet solution was measured at 590 nm using a plate reader (Biotech ELx800, UK).