Non adherent 96 well plate

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).

To examine sphere forming capacity in response to IL-7, the sphere formation assay was performed as described previously⁶⁹ . Cells were seeded on nonadherent 96-well plates (Corning) at 2 × 10²/100 μL/well (n = 20) with or without IL-7 and maintained for one week. The medium was not changed or added so as to disturb the formation of sphere. Visible spheres (>100 μm in diameter) were counted under a microscope (×10) after 7-day incubation. For serial passages of the sphere, spheres were dissociated into single cells using enzymatic digestion with trypsin-EDTA (Life Technologies). The total number of spheres formed from 20 wells was counted each experiment, and the average for three independent experiments was calculated.