Mammocult human medium kit

Cells derived from MDA-MB-231 and MCF-7 cell lines were seeded in 6-well low attachment suspension culture plates (Corning^® Costar^® Ultra-Low Attachment Multiple Well Plate, Thermo Fisher Scientific, Waltham, MA, USA) at a density of 3.5 × 10⁴ viable cells/well. Cells were grown in 2 mL MammoCult Medium Human Kit, enriched with Proliferation Supplement 0.1 mg/mL, Heparin Solution 4 µg/mL, Hydrocortisone Stock Solution 0.48 µg/mL (all StemCell Technologies, Vancouver, BC, Canada) and antibiotics (1% penicillin/streptomycin, Sigma-Aldrich, Steinheim, Germany) for control, and with addition of 2 µM 1 in enriched media for tested cells. Mammospheres larger than 50 μm were counted after 7 days of incubation using a Motic AE31E Inverted Microscope (Thermo Fisher Scientific, Waltham, MA, USA).

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.

Drug-resistant cell line MCF-7/ADR was cultured using DMEM (Thermo Scientific Hyclone, MA, USA) supplied with 10% fetal bovine serum and 100 U/mL penicillin and 100 μg/mL streptomycin at 37 °C and 5% CO₂. To maintain a highly drug-resistant cell population, MCF-7/ADR cells were periodically reselected by growing them in the presence of 1000 ng/mL Adriamycin. Experiments were performed using the cells incubated without DOX for 48 h. CD24- and CD44-microbeads antibodies (Miltenyi Biotec, Germany) were used for cell sorting of Breast Cancer Stem Cells (BCSCs) [15 (link)]. Briefly, 10⁷ total MCF-7/ADR cells were incubated with the above antibodies on ice for 40 min. After washing with cold PBS, CD44 + CD24 −/low BCSCs named MCF-7/ADR CSCs were purified from MCF-7/ADR cell lines. The characteristics of MCF-7/ADR CSCs were regularly detected by flow cytometry and maintained into ultra-low attachment six well plates (Corning, New York, USA) in MammoCult™ Human Medium Kit (Stem cell technologies, Vancouver, Canada) according to manufacturer’s guideline [16 (link)].

Cells (2.5–5×10³) were seeded per well in 6-well ultra-low attachment culture plates (Corning) using the MammoCult Human Medium Kit (Stemcell Technologies). The mammospheres were (i) treated with vehicle or 500-ng/mL DOX (doxycycline), (ii) left untreated or treated with GO-203, (iii) treated with vehicle or carboplatin, and (iv) treated with vehicle or olaparib. Mammospheres with diameters >100 μm were counted in triplicate under an inverted microscope.