6 well ultralow attachment culture plate

Single-cell suspensions were cultured in MammoCult Human MediumKit (Stemcell Technologies, Cambridge, MA, USA) at a density of 5,000 cells per well of a 6-well ultralow attachment culture plate (Corning) for 10 days as described [27 (link)]. Tumorspheres with a diameter >50 microns were counted under an inverted microscope in triplicate wells.

Single-cell suspensions were cultured in MammoCult™ Human Medium Kit (Stem Cell Technologies) at a density of 2,000 to 10,000 cells per well of a 6-well ultralow attachment culture plate (Corning CoStar). For first generation M1 culturing, cells were grown with replenishment of the medium twice over 7 days. For second M2 generation culturing, M1 mammospheres were harvested, incubated with trypsin for 3 min at 37°C, and mechanically dispersed by gentle pipetting. Single cells were confirmed under a microscope, counted and resuspended in fresh MammoCult™ medium. Mammospheres were visualized using a Nikon inverted TE2000 microscope and scored as positive when ≥50 μm in size. Sphere forming efficiency (SFE) was calculated by dividing the number of mammospheres by the number of suspended cells.

Cells were plated in a 6-well ultra-low attachment culture plate (Corning Incorporated, Corning, NY, USA) at a density of 1 × 10³ cells/well and cultured in serum-free DMEM/F12 cell medium (Gibco-Invitrogen) supplemented with 20 ng/ml epidermal growth factor (EGF, ThermoFisher, Waltham, MA, USA), 20 ng/ml basic fibroblast growth factor (bFGF, ThermoFisher), 4 μg/ml heparin (Sigma-Aldrich), and 0.4 μg/ml B27 (ThermoFishher) and chased every 4 days until day 11. The growth characteristics of spheroids were observed and the diameter of a micro-spheroid was determined as indicated above.