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. 2017 Apr 20;7(8):e2233.
doi: 10.21769/BioProtoc.2233.

Melanoma Stem Cell Sphere Formation Assay

Alessandra Tuccitto  1 , Valeria Beretta  1 , Francesca Rini  1 , Chiara Castelli  1 , Michela Perego  2
Affiliations

Melanoma Stem Cell Sphere Formation Assay

Alessandra Tuccitto et al. Bio Protoc. .

Abstract

Self-renewal is the ability of cells to replicate themselves at every cell cycle. Throughout self-renewal in normal tissue homeostasis, stem cell number is maintained constant throughout life. Cancer stem cells (CSCs) share this ability with normal tissue stem cells and the sphere formation assay (SFA) is the gold standard assay to assess stem cells (or cancer stem cells) self-renewal potential in vitro. When single cells are plated at low density in stem cell culture medium, only the cells endowed with self-renewal are able to grow in tridimensional clusters usually named spheres. In the recent years, SFA has also been used to test the effect of several drugs, chemical and natural compounds or microenviromental components on stem cells self-renewal capacity. Here we will illustrate a detailed protocol to assess self-renewal of human melanoma stem cells, growing as melanospheres.

Keywords: Cancer stem cells; Melanoma; Melanospheres; Self-renewal; Stem cell medium.

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Figures

Figure 1.
Figure 1.. Melanoma sphere formation.
A. Single cell melanospheres plated in SCM; B. Doublets appearing when cells start dividing; C. Triplets formed during cell proliferation; D. Fully formed melanospheres. Total magnification 200x (A-D), scale bar = 20 µm.
Figure 2.
Figure 2.. Non-melanosphere clusters.
Example of non-spherical cluster that could be found in SFA. Those clusters could not be counted as spheres. Total magnification 200x, scale bar = 20 µm.
See this image and copyright information in PMC

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