A high-throughput workflow for assessing self-renewal using colony formation assays.

The colony formation assay (CFA) is a widely used method to assess the self-renewal capacity of cancer cells and evaluate how this property is affected by drug treatment. This protocol presents a streamlined, high-throughput CFA workflow optimized for T-cell acute lymphoblastic leukemia (T-ALL), making it suitable for large-scale drug screening projects. Colonies are grown in a methylcellulose-based 3D matrix and quantified using an automated analysis pipeline, allowing robust estimation of colony number and size. This cost-effective approach provides a scalable platform for identifying compounds that impair self-renewal, facilitating prioritization of hits for validation in more complex in vivo models.