Colcount colony counter

Cells were plated at 1,000 cells per well in a 6-well plate and media was replaced every two days for 12 days total. For trastuzumab treatments, trastuzumab was added to media at 10 µg/ml and media was replaced with fresh control or trastuzumab containing media every two days during the experiment. Colonies were fixed in 100% methanol and stained with crystal violet. Colony number and diameter were calculated using a ColCount Colony Counter (Oxford Optronix) and data was analyzed using the supplied statistical software.

After irradiation of the cells in culture flasks, cells were trypsinized and seeded in triplicate in six-well plates at low densities. Fourteen days after seeding, cells were fixed with 6% glutaraldehyde and 0.5% crystal violet (Sigma). Colonies of more than 50 cells were counted with a ColCount colony counter (Oxford Optronix, Oxford, UK). To calculate the surviving fraction (SF), the following formula was used:
The survival curve was fitted to the LQ model using the following formula:
in this formula α and β are radiation sensitivity parameters, D is the irradiation dose. At least three independent experiments were performed with three replicates per experiment. Due to experimental limitations, we were not able to obtain a larger number of control samples (compared to irradiated samples). Therefore, it will not be possible to detect slight changes in the survival of cells at lower doses such as 0.5 Gy. The RBE was calculated at a 10% survival by dividing the dose of X-rays at SF₁₀ by the corresponding dose of proton or carbon ions at SF₁₀. In order to determine the effect of the drug the sensitizer enhancingratio (SER₁₀) was calculated by taking the ratio of the doses to reach 10% survival for control cells over the treated cells.

Cells were seeded in a 6-well plate at 7.5–30 × 10⁴ cells per well and the next day drug or solvent was added. After 72 h incubation with drug, cells were trypsinized and plated in triplicate at low density in 6-well plates. When cells were attached (after 4 h), they were irradiated with 2, 4 and 6 Gy using a Baltograph (Balteau NDT, Hermalle-sous-argenteau, Belgium) or mock irradiated. Medium was changed 16 hours post-irradiation. After 11–21 days, cells were fixed with 2.5% glutaraldehyde in PBS and stained with 0.4% crystal violet. The colonies containing 50 cells or more were counted with ColCount colony counter (Oxford Optronix, Oxford, UK). Survival fractions were calculated after normalizing for drug-induced toxicity. Dose-enhancement factor (DEF0.5) was calculated as the ratio of the dose needed for the control cells to the dose needed for the treated cells to reach a survival fraction of 0.5.

Clonogenic assays were performed in 10 cm tissue culture dishes (for RPE-1 cells, U2OS cells and derived clones). Cells (1000 per dish) were seeded in complete media (10 mL) and allowed to attach overnight, then treated with cisplatin or cisplatin plus inhibitor for the times stated. Following treatments, cells were allowed to grow and form colonies for 10 days. Colonies formed were stained with Coomassie R250 (Sigma) and counted on a COLCount Colony Counter (Oxford Optronix). All experiments represent the mean (±SEM) of at least three biological repeats of duplicate dishes/flasks for each treatment.

The survival of NSCLC cells, when challenged with cisplatin, was measured using the clonogenic survival assay. Cells were seeded at optimal cell densities and allowed to adhere overnight at 37 °C. Cells were treated with increasing concentrations of cisplatin (0–10 μM) for 72 hr, alone or in combination with ATRA (5 μM) or retinol (1 μM), after which time culture media was removed and replaced with fresh treatment-free media and re-incubated for 10 days. Colonies were fixed and stained with 25% (v/v) methanol, 0.05% (w/v) crystal violet for 30 mins. Residual stain was removed by rinsing wells gently with water. Colonies were counted using the ColCount™ colony counter (Oxford Optronix Ltd, Oxford, UK). Plating efficiencies (PE) were calculated using the formula: PE = Number of colonies/Number of cells seeded. The percentage surviving fraction (SF) was calculated using the formula: SF = (PE treated colonies/PE untreated) × 100.