Ultra low adherence 24 well plates

For colony-formation assays, isolated luminal cells were plated on irradiated 3T3 cell feeders in 24-well plates at a density of 500 cells per well and cultured in DMEM/F12 medium supplemented with 10% FBS, 5 μg/mL insulin (Sigma-Aldrich), 10 ng/mL EGF (Invitrogen, Life Technologies), and 100 ng/ml cholera toxin (ICN Biochemicals) for 7–8 days, as previously described [8 (link), 23 (link)].
For mammosphere-formation assays, 5000 isolated luminal cells were seeded on ultralow-adherence 24-well plates (Corning) in DMEM/F12 medium supplemented with 2% B27 (Stem Cell Technologies), 20 ng/mL EGF, 20 ng/mL bFGF (GIBCO, Life Technologies), 4 μg/mL heparin (Sigma-Aldrich), 10 μg/mL insulin, and 2% growth-factor-reduced Matrigel (BD Biosciences) for 10–12 days. For second-generation sphere assays, mammospheres were dissociated with 0.05% trypsin (Gibco, Life technologies) and reseeded as described above. When specified, cells were treated with 25–50 ng/ml recombinant mouse HGF (R&D Systems Europe) every 2 days, as described elsewhere [8 (link)]. ImageJ software (NIH) was used to count colonies and mammospheres and quantify their size in pixels.

MCF-7 and MDA-MB-231 cells were cultured in 6-well plates and exposed to drugs as mentioned above. The cells were collected and furthered cultured in ultra-low adherence 24-well plates (Corning, MA, USA) with 1 ml stem cell culture medium (serum-free DMEM-F12 supplemented with B27 (Invitrogen), 20 ng/ml epidermal growth factor (Peprotech), 10 ng/ml basic fibroblasts growth factor (Peprotech), 10 μg/ml insulin (Sigma)) at a density of 10,000 cells/well. After 7–10 days culture, the mammospheres were photographed.

Cells were pre-treated with IgG3, anti-APO-1, LzCD95L, IFNβ, or received no treatment for 6 days. Cell suspensions were passed through a 40 μm sterile cell strainer (Fisher Scientific) to obtain single cell suspension. The strained cell suspension was diluted in Mammocult media and seeded at 5000 cell/well in triplicate in ultra-low adherence 24-well plates (Corning). The cells were cultured in Mammocult media (Cell Stem Technology; prepared according to manufacturer’s instructions) supplemented with 4 μg/ml Heparin, 0.5 μg/ml hydrocortisone and 10% Mammocult Proliferation Supplement. Spheres were counted using a light microscope 6 days after seeding.

DPSC attachment was monitored by fluorescence microscopy. Cells were seeded onto the surface of sterilized pSi particles at a cell density of 5 × 10⁴ cells/mL. Cells were incubated for 24 h at 37 °C with 5% CO₂ in a humidified incubator, in ultra-low adherence 24 well plates (Corning, NY, USA) that inhibit cell attachment on the tissue culture plate, allowing DPSC to attach only to the pSi particles. After the incubation time, the cells were fixed in 2.5% glutaraldehyde and stained for actin cytoskeleton and nuclei. Cells were permeabilized with 0.5% Triton X-100 in PBS at 4 °C for 15 min, incubated for 1 h with TRITC-labeled phalloidin (1:200) at 37 °C in the dark, then with Hoechst 33342 (Life Technologies, Carlsbad, CA, USA) for 10 min at room temperature, and washed twice with deionized water. Samples were observed under fluorescence microscopy (Nikon TE2000, Nikon Instruments, Amsterdam, The Netherlands) at an excitation wavelength of 360 nm for Hoechst staining and 540 nm for phalloidin labeling.