MDA-MB-435 and MDA-MB-231 cells were grown in 100 mm dishes in DMEM/F12 medium supplemented with 10% FBS to 60% confluence. Cells were then washed twice in PBS, harvested and counted. Cells (5×103) in 0.1 mL complete Mammocult™ medium (STEMCELL Technologies) were seeded onto ultra-low adherent six-well plates (STEMCELL Technologies) containing 1.9 mL/well of complete Mammocult medium supplemented with APR-246 at the final concentrations indicated. For controls, an equal volume of PBS (vehicle) was added to the medium. Incubations were carried out in triplicate. Every 48 hrs, cells were retreated with additional 1 mL of fresh APR-246 (or vehicle) in complete Mammocult medium. Images of mammospheres were captured by EVOS light microscopy (10x objective) (Waltham, MA, USA) on days 2, 4, and 6 of treatment. Mammospheres≥100 μm were quantified.
Ultra low adherent six well plates
Manufactured by STEMCELL
Sourced in United States
Ultra-low adherent six-well plates are laboratory consumables designed for cell culture applications. They feature a surface treatment that minimizes cell attachment, promoting the formation of 3D cell aggregates or spheroids. These plates are suitable for a variety of cell types and research applications that require low-adherence culture conditions.
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2 protocols using ultra low adherent six well plates
1
Mammosphere Formation Assay with APR-246
2
Mammosphere Formation and Treatment
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Mammosphere formation was performed using MammoCult media (Stemcell Technologies, Cambridge, MA, USA) supplemented with 4 µg/mL heparin and 0.48 µg/mL hydrocortisone, according to the manufacturer’s instructions. In brief, 4 × 104 control and NQO1 stable cells were seeded into ultralow adherent six-well plates (Stemcell Technologies, Cambridge, MA, USA)) and incubated with MammoCult media for one or two weeks. Then, bL (2 µM) was added for 24 h, and the cell morphology was observed under a hybrid microscope (Echo, San Diego, CA, USA).
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