Ultralow attachment 100 mm plates

For the limiting dilation assays (LDA), MDA-MB-231 and BT-549 primary tumour spheres (TS), obtained from 21-day old spheroids, were mechanically disaggregated and counted before being seeded in non-adherent 96-well plates (Corning). Next, serial dilutions were performed to achieve 200 cells/well down to 1 cell/well. Spheroid formation was monitored until day 21 by determining the number of TS per well.
For the secondary and tertiary TS formation assays, primary TS were mechanically dissociated after which 200.000 cells were cultured in ultralow attachment 100 mm plates (Falcon) in the presence of JQ1 (200 nM) for 3 days. Next, the number of secondary TS per plate were determined. Secondary TS were again dissociated and left in the presence of the drug until day 6, at which the number of tertiary TS per plate was determined. TS pictures were taken at the two time points using an inverted microscope (Nikon).

MDA-MB-231-derived tumorspheres (TS) were mechanically dissociated by up and down pipetting and 200.000 cells cultured in ultralow attachment 100 mm plates (Falcon) in the presence of JQ1 (200 nM), Volasertib (10 nM) or combination of both. The following day, TS were again dissociated. At 72 hours, newly formed TS were blindly counted on 25 random fields of view (FOV). TS/FOV was calculated from 3 independent experiments. Two-ways Student's test was used for the statistical analyses.