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Utilizing three-dimensional in vitro sphere culture conditions provide a better representation of tumor growth in vivo than the more common monolayer cultures. We describe how to perform primary and secondary sphere assays, used for the enrichment and self-renewability studies of melanoma\/melanocyte stem-like cells. Spheres are generated by growing melanoma cells at low density in nonadherent conditions with stem cell media. We provide protocols for preparing inexpensive and versatile polyHEMA-coated plates, setting up primary and secondary sphere assays in almost any tissue culture format and quantification methods using standard inverted microscopy. Our protocol is easily adaptable to laboratories with basic cell culture capabilities, without the need for expensive fluidic instruments.<\/jats:p>","DOI":"10.1007\/978-1-0716-1205-7_14","type":"book-chapter","created":{"date-parts":[[2021,3,11]],"date-time":"2021-03-11T12:03:16Z","timestamp":1615464196000},"page":"185-199","update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":16,"title":["Enrichment of Melanoma Stem-Like Cells via Sphere Assays"],"prefix":"10.1007","author":[{"given":"Nabanita","family":"Mukherjee","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Karoline A.","family":"Lambert","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"David A.","family":"Norris","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yiqun G.","family":"Shellman","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"297","published-online":{"date-parts":[[2021,3,12]]},"reference":[{"issue":"5","key":"14_CR1","doi-asserted-by":"publisher","first-page":"486","DOI":"10.1016\/j.stem.2011.04.007","volume":"8","author":"E Pastrana","year":"2011","unstructured":"Pastrana E, Silva-Vargas V, Doetsch F (2011) Eyes wide open: a critical review of sphere-formation as an assay for stem cells. 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