An improved method for monitoring cell death in cell suspension and leaf disc assays using evans blue

Abstract

Cell viability or cell death is an important variable to monitor in many studies of host/pathogen interactions. However for studies that focus on events within the first few hours of the interaction, many of the viability assays currently being used are either too laborious and time consuming or measure the cell's temporary metabolic state rather than irreversible cell death. Evans blue has proven over the years to be a dependable stain for microscopic determination of cell death. We have used this stain to develop a spectrophotometric procedure that allows rapid, reproducible quantification of the stain retained by dead cells. This spectrophotometric procedure was used to compare plant/bacteria interactions involving either soybean/Pseudomonas syringae pv. glycinea or tobacco/P. syringae pv. syringae. Relative increases in cell death during these interactions in suspension cell systems were measured by both the spectrophotometric and microscopic technique and found to be similar. The spectrophotometric procedure was also adapted for leaf disc assays.