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This article provides a set of protocols for generating uniform spheroids with a thin sheet of basement membrane for studying the initial invasion of mammary epithelial cells into a surrounding collagen-rich extracellular matrix. Details are provided for generating 3D spheroids with a basement membrane, polymerizing collagen I, embedding the spheroids in the 3D collagen gel, and immunostaining the spheroids for invasion studies. Published 2020. U.S. Government. Basic Protocol 1 Growth of uniformly sized tumor spheroids with an encapsula