Dead protoplasts are stained red by:
Dead protoplasts are stained red by propidium iodide PI nucleic acid intercalating fluorescent dye that distinguishes membrane compromised cells. PI is cationic molecule unable to cross intact lipid bilayer of viable protoplasts due to charge and size exclusion, so viable population excludes dye remaining unstained. In dead cells membrane integrity lost through enzymatic damage or physical stress, PI diffuses entering cytoplasm nucleus where it intercalates between double stranded DNA and RNA base pairs with strong affinity, enhancing red fluorescence emission at 617 nm when excited green light 535 nm. Under fluorescence microscope dead protoplasts show bright red nucleus clearly differentiated from viable green FDA positive cells in dual viability test. This assay allows rapid quantitative assessment of protoplast isolation success and cytotoxicity of fusogen treatments. Compared to colorimetric dyes like Evans blue which stains dead cells blue but requires bright field observation, PI provides fluorescent contrast compatible with flow cytometric analysis sorting live cells for downstream culture fusion transformation. Maintaining membrane integrity crucial because only viable protoplasts can regenerate wall divide form microcallus and participate in heterokaryon formation during somatic hybridization procedures.
Ref: Jones 1987 PI dead staining; Molecular Probes Handbook viability dyes.