Mammocult basal medium human

Fluorescently tagged MDA-MB-231 cells in culture or freshly resected from tumors that were processed (enzymatic digestion in 2 mg/mL collagenase for 1 h at 37 °C with orbital shaking at 160 rpm) were passed through a cell strainer (70 μm) and plated at 2,000 cells/well in 12-well plates previously coated with polyHema (12 g/L in 95% EtOH) and air-dried for 48 h, with 2 mL of mammosphere formation media per well (MammoCult basal medium (human) containing 4 μg/mL heparin, and 0.48 μg/mL hydrocortisone) (STEMCELL Technologies; Vancouver, BC, Canada). Fluorescent images were captured using a Cytation 5 (BioTek Instruments) equipped with an Olympus–UPLFLN 4XPh phase objective. A montage of the entire well was constructed by stitching individual images. The total number and average size of each mammosphere were then calculated using Gen5 software (BioTek instruments) automated counting algorithm.

Single cells were individually seeded through cell sorting (BD FACS Jazz) in 96 well Ultra-low attachment plates containing MammoCult™ Basal medium (Human) (Stem Cell Tech) supplied with hydrocortisone 0.48 mg/mL, Heparin solution 0.2% and 10% MammoCult™ Proliferation Supplement (Human). After 21 days, primary tumorspheres formed were measured both in size and number using inverted microscope (Olympus CKX41).

A total of 5x10³ cells/mL/well were seeded in Ultra-low Attachment Plates containing MammoCult™ Basal Medium (Human) (Stem Cell Tech) supplied with 0.48 mg/mL hydrocortisone, 0.2% heparin solution and 10% MammoCultTM Proliferation Supplement (Human). After 4 days, primary tumorspheres were measured, both in size and in number, using an inverted microscope (Olympus CKX41).

Freshly resected MDA-MB-231 tumors were subjected to physical dissociation followed by enzymatic digestion (collagenase 2 mg/mL (Sigma-Aldrich), BSA 2 mg/mL (Gemini Bioproducts)) for 1 h at 37 °C at 160 RPM. Cells were then passed through a cell strainer (0.7 μM), and 10,000 cells/well were transferred to a 6-well plate previously coated with polyHEMA (12 g/L in 95% EtOH) and air-dried for 48 h with 2 mL of mammosphere formation media per well (MammoCult basal medium (human) with 4 ug/mL heparin, and 0.48 ug/mL hydrocortisone) (STEMCELL Technologies; Vancouver, BC, Canada).

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.

Sorted cells (1 × 10³ cells) were seeded in a 24-well ultra-low adherent plate (Corning) in 0.5 mL of mixed medium to perform sphere formation detection. The medium contained 32% MethoCult medium, 20% MammoCult basal human medium with a final concentration of 2% MammoCult proliferation supplements and 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 mg/mL insulin, all obtained from STEMCELL Technologies. The cells were cultured at 37 °C in a 1% O₂ and 5% CO₂ humidified atmosphere for 14 d. The number of tumor spheres was counted under microscope.