Quantification of Membrane Damage/Cell Death Using Evan's Blue Staining Technique

Membrane damage is a hallmark of both biotic and abiotic stress responses. The membrane determines the ability of a cell to sustain altered environmental conditions and hence can be used as a biomarker to assess stress-induced cell damage or death. We present an easy, quick, cost-effective, staining and spectrophotometric method to assess membrane stability of plant cells. In this method, Evan's blue, an azo dye, is used to assay for cell viability. More specifically, Evan's blue dye can penetrate through ruptured or destabilized membranes and stain cells. Thus, when plant cells are subjected to stress that compromises membrane integrity, the number of cells that are permeated by Evan's blue dye will be increased compared to control cells that are not stressed. In contrast, live, healthy cells that are capable of maintaining membrane integrity do not take up Evan's blue dye. Cells that have taken up Evan's blue dye will have an accumulation of a blue protoplasmic stain and these stained cells can be qualitatively documented under bright field microscopy with or without the use of a camera. Furthermore, the dye can be extracted from cells that are stained by Evan's blue dye and can be quantified spectrophotometrically. Using this analysis, the accumulation of dye in positively-stained cells correlates with the extent of cell membrane damage and thus the amount of cells that are stained with Evan's blue dye under various conditions can be used as an indicator of cellular stress.