Mammocult proliferation supplement

Cells were transfected with miR‐206 mimics or negative control, incubated (24 hr), and diluted in MammoCult human medium, supplemented with MammoCult proliferation supplements (Stemcell Technologies, BC, Canada) in a 1:10 ratio, 4 μg/ml (0.0004%) heparin solution, 0.48 μg/ml hydrocortisone, and 1% PEST. Cells were seeded in low‐adherent six‐well plates (10,000 cells/well) with 2 ml of final volume. Cells were incubated (5% CO₂; 37°C) for 7 days to allow primary mammospheres to form. Plates were scanned in 2400 dpi resolution using GelCount, version 1.1.2.0 (Oxford Optronix Ltd, Milton, UK). For secondary cultures, cells from the primary culture were rinsed twice with PBS, brought to single‐cell suspension using Trypsin–EDTA treatment, and mammosphere assays conducted following the miR‐206 mimics or control transfection as described above, but with 5000 cells/well. Plates were scanned and images of the mammospheres were analyzed as above. For primary mammosphere assays, three experiment were performed using HC11 cells of three different passages, each in technical triplicates. For secondary mammosphere assays, three experiment were performed using HC11 cells of three different passages, one in technical triplicates and two without technical replicates.

Tumorsphere-forming analysis was performed as described by Dontu et al (Dontu et al, 2003 (link)). A549 and HCC1806 cells were pretreated with (1) P2S or P2 (10 μM) and (2) PBS or IAA (500 μM) for 2 days, and then were dissociated into single-cell suspensions and plated in 96 wells of ultra-low attachment plates (cat. No. 7007, Corning Life Sciences) at 300 to 500 (A549 serials) and 1000–2000 (HCC1806 serials) cells/well respectively in MammoCult™ medium (cat. No. 05621, STEMCELL Technologies) supplemented with MammoCult™ proliferation supplements (cat. No. 05622, STEMCELL Technologies), 4 μg/mL heparin (cat. No. 07980, STEMCELL Technologies), 0.48 μg/mL hydrocortisone (cat. No. H0888, Merck), 100 U/mL penicillin, 100 μg/mL streptomycin, and 1% methylcellulose (cat. No.M7027, Merck) After 2 weeks, the number of spheres (diameter >100 μm) of each well was calculated.

Untreated and p53 SMWC-treated human carcinoma cells were planted in triplicate wells (1 × 10³ cells/well) in 24-well Ultra-Low attachment plates (Corning Incorporated) with sphere formation medium (500 μL of mixed medium containing 32% MethoCult medium / 20% MammoCult basal human medium (final concentration of 2% MammoCult proliferation supplements (Stem Cell Technologies), including 48% DMEM supplemented with final concentrations of 100 pg/mL EGF, 50 ng/mL bFGF, 5 ng/mL stem cell factor, 1 μM hydrocortisone, and 5 μg/mL insulin at 37°C in a 1% O₂ and 5% CO₂ humidified atmosphere for 12 d. The total sphere number was the sum of the number of the spheres counted in 6 random fields of each well using a Zeiss Inverted Fluorescence Microscope at 100X magnification.