Use of Propidium Iodide
Propidium Iodide (PI) is a nuclear dye, intercalating into DNA (and also into RNA). Similar to DAPI and Höchst, it is not penetrating the cell membrane and therefore will not label the nuclei of living cells. This is often used in mortality assays with animal cells, for instance when apoptosis is analysed by cell sorting. Propidium Iodide is excited by green light and then yields a red fluorescence. For this reason, it can be used as nuclear dye in confocal laser microscopy instead of DAPI / Höchst, because a normal ArKr laser is sufficient.
- absorption at 535 nm, emission at 617 nm after binding (unbound 570 nm and 600 nm)
- stochiometry 1 molecule PI on 4-5 bases
- upon binding, fluorescence enhanced 30-fold
- molecular weight 668
- stock solution 1.5 mM in water, working solution 0.5-1.5 µM
- storage light protected, at 4°C 6 months, at -20°C longer
- not membrane permeable
- mutagenic, cancerogenic, teratogenic!
Applications in Plants
- labelling apoptotic cells (nuclei labelled)
- general stain of cell walls for whole-mount confocal preparations of roots
- target of binding are pectins, so it can be used to quantify pectins in vivo
- PI also intercalates into RNA, therefore there is some cytoplasmic background (seldom a problem in plant cells)
- if there is high cytoplasmic background, RNase digestion is recommended
- staining of living cells with PI: watch immediately, prolonged staining perforates the membrane.