Olaparib

To characterize how the tumor cells grew in the absence of treatment and when exposed continuously to different drug concentrations, we seeded cells in a 48 well flat-bottom plate (Costar Corning) and left them to attach overnight in 200µL culture medium. Subsequently, we aspirated the medium and replaced it with treated growth medium, containing 0, 1, 10, 25, 50 or 100µM Olaparib (AstraZeneca), and monitored their growth for 9 days. We carried out two versions of this assay: i) a “low-density” version in which we seeded cells at 5,000 cells per well, and ii) a “high-density” version in which each well started with 60,000 cells. In each case, 3 replicates were performed for each experimental condition. During the experiment the medium was changed every 3 days. We also tested changing the medium daily but found that this did not significantly change the growth dynamics (not shown).
To prepare the treated medium, we first created a stock containing 100µM of drug by dissolving Olaparib (AstraZeneca) in 1mL Dimethyl sulfoxide (DMSO), filtering the solution using a 0.22nm syringe filter, and dissolving it in our regular culture medium. Next, we diluted this maximum tolerated dose (MTD) stock with normal culture medium to obtain batches with 1–50µM Olaparib. We verified that the DMSO did not adversely impact the cells’ growth dynamics (not shown).