Ultra low adherent plate

After the cells were transfected with miR-21-5p mimic for 48 hours or miR-21-5p inhibitor for 24 hours, 2.5 × 10⁵ cells/well were cultured in DMEM/F12 (1:1) with B27 (Invitrogen), 40 ng/ml FGE-2, 20 ng/ml EGF (Peprotech, Rocky Hill, NJ) in a 6-well ultra-low adherent plate (Corning, Corning, NY) for 7 days. Spheres >50 μm in diameter were counted, digested with trypsin, and resuspended. The total number of cells in the single-cell suspension was counted; the average number of cells in each sphere was calculated using the total cell number divided by the number of spheres.

Details of sphere formation by cancer cell lines were described previously [58 (link)]. Briefly, the HER2+/− breast cancer cell lines were grown in MEM medium supplied with 10% FBS, the sub-cultures were depleted from the serum-containing medium and 2-5 × 10⁶ cells were cultured on ultra-low adherent plate (Corning, MA) in MEM supplemented with epidermal growth factor (EGF, 20 ng/ml), basic fibroblast growth factor (FGF, 10 ng/ml), and 1–5% B27 supplements (Life Technologies, CA). The formed spheres were enzymatically dissected into single cell suspension and further subjected to at least two cycles of sphere formation. Total RNA was reversed transcribed using a high capacity cDNA reverse transcription kit (Applied Biosystems). The cDNA was then used for expression analysis of ALDH1, SOX2, NANOG and OCT3/4 employing quantitative real time PCR (Power SYBR Green PCR Master Mix, Applied biosystems). Primer sets for targeting these genes were designed using primer design software from Integrated DNA Technology and were confirmed by “BLASTing” each primer sequence against the human genome using NCBI and UCSC Genome Browser (Table S4). All Cycle Threshold C_t values of the genes were normalized to housekeeping gene, i.e., GAPDH as described

The individual cells (10⁴ cells/mL) were cultured in serum-free Dulbecco's modified Eagle medium/F-12 containing B27 (Invitrogen), 20 ng/mL epidermal growth factor (BD Bioscience), and 10 ng/mL basic fibroblast growth factor (BD Bioscience). After culture for 7 to 10 days in the ultra-low adherent plate (Corning, NY, USA) under conventional conditions, the spheres were photographed, and tumor spheres larger than 50 μm in diameter were counted.

Mammospheres were seeded and cultured as previously described [16 (link)]. Briefly, cells were trypsinized and passed through a 40 μm cell strainer (BD, Franklin Lakes, New Jersey) and seeded into ultra-low adherent plates (Corning) in Mammocult media (StemCell Technologies, Vancouver, BC, Canada). For subsequent RNA extractions, mammospheres were isolated and collected by centrifugation as per manufacturer’s protocol.

Mammospheres were seeded and cultured as previously described [67 (link)]. Briefly, cells were trypsinized and passed through a 40 μm cell strainer (BD, Franklin Lakes, New Jersey, USA) and seeded into ultra-low adherent plates (Corning, NY, USA) in Mammocult media (StemCell Technologies, Vancouver, BC, Canada) as per manufacturer's instructions. Mammosphere larger than 60 μm were counted 5-7 days after seeding. Limiting dilution assay has been used as a gold standard for the assessment of cancer stem cells [33 (link), 34 (link)]. To perform these experiments, cells were seeded in 96-well low-adherent plate (Corning, NY, USA) at 10 limiting dilutions ranging from 1 to 400 cells. Each dilution had 6 replicates, and each well was scored for presence or absence of mammosphere after 5-7 days. Data were analyzed using the Extreme Limiting Dilition Analysis (ELDA) software for three independent experiments [68 (link)].