What is AO PI staining?
Acridine orange (AO) and propidium iodide (PI) are nuclear staining (nucleic acid binding) dyes. AO is permeable to both live and dead cells and stains all nucleated cells to generate green fluorescence. PI enters dead cells with compromised membranes and stains all dead nucleated cells to generate red fluorescence.
Does DAPI only stain dead cells?
DAPI and PI only inefficiently pass through an intact cell membrane and, therefore, preferentially stain dead cells. TMRM is a cell-permeable fluorescent dye that is sequestered to active mitochondria, and hence labels live cells.
What is are the limitation s of using PI stain to determine the DNA content in cell?
PI has two disadvantages; it stains all double stranded nucleic acids so the cells have to be incubated with RNase to remove any double stranded RNA and the dye is excluded by the plasma membrane so that the cells have to fixed or permeabilised before adding the dye.
What is AO dye?
Acridine Orange is a cell-permeant nucleic acid binding dye that emits green fluorescence when bound to dsDNA and red fluorescence when bound to ssDNA or RNA.
Why is propidium iodide used to identify cells that are both proliferating and have undergone necrotic death?
Since propidium iodide is not permeant to live cells, it is also commonly used to detect dead cells in a population. PI binds to DNA by intercalating between the bases with little or no sequence preference.
Does DAPI only emit UV light?
When bound to double-stranded DNA, DAPI has an absorption maximum at a wavelength of 358 nm (ultraviolet) and its emission maximum is at 461 nm (blue). Therefore, for fluorescence microscopy, DAPI is excited with ultraviolet light and is detected through a blue/cyan filter. The emission peak is fairly broad.
Is DAPI hydrophobic or hydrophilic?
Indeed, as DAPI is hydrophobic it confers this behaviour on the DNA and thus restricts the presence of water molecules.
What stages of apoptosis can PI detect?
Therefore, by staining cells with a combination of annexin V–FITC and PI, it is possible to detect unaffected, non-apoptotic cells (annexin V–FITC negative/PI negative), early apoptotic cells (annexin V–FITC positive/PI negative), and late apoptotic (“necrotic stage” of apoptosis) as well as necrotic cells (PI positive …
Does acridine orange stain DNA?
STAINING TECHNIQUES Acridine orange is a compound that differentially stains DNA (yellow-green) and RNA (bright red).