Quantifying loss of cell viability or adhesion using methylene blue

Methylene blue is a versatile dye used in cell biology to quantify cell adhesion and indirectly assess cell death. Unlike viability assays that rely on metabolic activity or membrane integrity, methylene blue binds to cellular proteins, making it particularly effective for staining adherent cells. This property is the basis of the assay we describe here, which allows direct measurement of cell detachment, a hallmark of various cell death processes, including apoptosis and anoikis. Using a simple staining and washing procedure, methylene blue selectively labels cells that remain attached to the culture surface after detached or dying cells have been removed. After washing, the retained dye can then be eluted and quantified spectrophotometrically, providing a reliable and cost-effective method for evaluating cytotoxicity, drug responses, and adhesion dynamics. This chapter outlines the principles, advantages, and practical applications of methylene blue staining in cell-based assays, highlighting its role as a powerful tool for studying cell survival, or detachment in both basic and applied research.