DNA diffusion assay is a simple, sensitive and reliable technique which allows the assessment of programmed cell death (PCD) or necrosis events based on nuclear morphology. It consists in isolating nuclei from plant material, which are then embedded in agarose and subjected to lysis in alkaline buffers. Under these conditions, and due to the presence of abundant alkali-labile sites in the DNA, small pieces of DNA diffuse in the agarose gel giving a specific halo appearance when stained with fluorescent dyes like DAPI (4',6-diamidino-2-phenylindole). Here, we describe an optimized protocol for DNA diffusion assay applied to different types of plant cells/tissues, indicating all the critical steps required for a successful experimental procedure.
Keywords: DNA diffusion; Necrosis; Nuclear morphology; Plant cells; Programmed cell death.