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

Background

Plants are sessile organisms that are exposed to a diverse array of stress factors. The membrane is made up of lipids and glycoproteins and act as a physical, protective barrier. The fluidity of the cell membrane is altered when the cell is exposed to stress such as heat. Oxidative stress can damage cell membranes. The reactive oxygen species (ROS) associated with oxidative stress can act on membrane lipids to decrease membrane stability. An established protocol to assess membrane stability, known as Sullivan’s method, quantifies the extent of electrolyte leakage from the membrane (Sullivan and Ross, 1979). This method is time-consuming, tedious and involves several steps. Additionally, since this method usually requires exposure of the tissue to high temperature (Initial electrolyte leakage and final electrolyte leakage after boiling at high temperature), this method cannot be used to assess the instantaneous damage to membranes in plants exposed to stress. We adapted a reliable Evan’s blue staining technique that has been used by many researchers to assess cell death or membrane damage (Smith et al., 1982; Oprisko et al., 1990; Vemanna et al., 2017) for instantly monitoring stress. Evan’s blue is an acidic, non-permitting exclusion dye which stains dead or damaged cells. The dye does not enter live cells with stable membranes (Gaff and Okong’O-Ogala, 1971). One advantage of this method is that it does not subject the tissue to high temperature. Though microscopic visualization is effective, large sample sizes make this type of analysis too time consuming (Baker and Mock, 1994). We have altered our method to analyze spectrophotometrically. Evan’s blue stain can be extracted from intact cells and analyze by spectrophotometer. Our method is highly reproducible and it can be adapted to large scale phenotyping of genotypes. The other membrane penetrating dye phenosafranin can also be used but some difficulties have been reported that it will not stain the cells without nuclei (ghost cell) and also the uptake is affected by pH (Baker and Mock, 1994).