DMEM/F-12 PLUS Basal Medium

Protocol tips

Upstream tips	Protocol tips	Downstream tips
You can develop chemoresistant cell lines	Seed 1.0 ×104 cells/well Use of 24-ultralow attachment plates Analysis of spheres with a diameter >40 μm	Collect spheres for other procedures, such as protein analysis

Upstream tips
You can develop chemoresistant cell lines
Protocol tips
Seed 1.0 ×104 cells/well Use of 24-ultralow attachment plates Analysis of spheres with a diameter >40 μm
Downstream tips
Collect spheres for other procedures, such as protein analysis

Publication protocol

BT-549 cells (1.0 ×104 cells/well) were plated in 24-ultralow attachment plates (Corning Incorporate) and grown in DMEM/F-12 serum-free medium (Sigma-Aldrich), supplemented with B27 (1:50, Invitrogen), 20 ng/ml basic fibroblast growth factor (bFGF, Sigma- Aldrich) and 20 ng/ml EGF. After 4 days, the size and number of spheres were analyzed under a phase-contrast microscopy (Leica DMI3000 B apparatus). All spheres with a diameter >40 μm were analyzed, and the number and size of spheres were quantitated in at least 10 fields per condition. At the end of treatments, mammospheres were collected by gentle centrifugation (1000 rpm) and RNA was recovered using TRIzol reagent and then processed for RT-qPCR.

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Manufacturer protocol

Download the product protocol from Sigma-Aldrich for DMEM/F-12 PLUS Basal Medium below.

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