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Related Experiment Video

Updated: Oct 19, 2025

Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines
10:51

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Published on: March 22, 2015

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Melanoma Stem Cell Sphere Formation Assay.

Alessandra Tuccitto1, Valeria Beretta1, Francesca Rini1

  • 1Department of Experimental Oncology and Molecular Medicine, Unit of Immunotherapy of Human Tumors, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.

Bio-Protocol
|September 20, 2021
PubMed
Summary

The sphere formation assay (SFA) is a key method for evaluating the self-renewal capacity of stem cells, including cancer stem cells. This protocol details how to use SFA for human melanoma stem cells, assessing their self-renewal potential in vitro.

Keywords:
Cancer stem cellsMelanomaMelanospheresSelf-renewalStem cell medium

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Area of Science:

  • Cell Biology
  • Cancer Research
  • Stem Cell Biology

Background:

  • Self-renewal is a fundamental property of stem cells, crucial for tissue homeostasis.
  • Cancer stem cells (CSCs) exhibit self-renewal, contributing to tumor growth and recurrence.
  • The sphere formation assay (SFA) is the standard in vitro method to assess stem cell self-renewal.

Purpose of the Study:

  • To provide a detailed protocol for assessing the self-renewal potential of human melanoma stem cells.
  • To demonstrate the application of the sphere formation assay (SFA) in the context of melanoma research.
  • To establish a reliable method for evaluating factors affecting melanoma stem cell self-renewal.

Main Methods:

  • Utilizing the sphere formation assay (SFA) for in vitro culture of single cells.
  • Plating cells at low density in specialized stem cell culture medium.
  • Culturing cells to form three-dimensional clusters known as spheres (melanospheres for melanoma).

Main Results:

  • The SFA successfully identifies and quantifies self-renewing human melanoma stem cells.
  • Melanosphere formation serves as a direct indicator of stem cell self-renewal capacity.
  • The protocol allows for the assessment of drug or compound effects on melanoma stem cell self-renewal.

Conclusions:

  • The sphere formation assay (SFA) is a robust and validated method for assessing human melanoma stem cell self-renewal.
  • This protocol facilitates research into melanoma stem cell biology and the development of targeted therapies.
  • SFA is instrumental in evaluating therapeutic interventions aimed at eliminating cancer stem cells.