Does PI bind to RNA?
Propidium iodide (PI) binds to DNA by intercalating between the bases with little or no sequence preference and with a stoichi- ometry of one dye per 4–5 base pairs of DNA. 1 PI also binds to RNA, necessitating treatment with nucleases to distinguish be- tween RNA and DNA staining.
Why is PI used in cell cycle analysis?
PROPIDIUM IODIDE: The most commonly used dye for DNA content/cell cycle analysis is PROPIDIUM IODIDE (PI). It can be used to stain whole cells or isolated nuclei. Since PI can also bind to double-stranded RNA, it is necessary to treat the cells with RNase for optimal DNA resolution.
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 …
How do you stain cells with PI?
Staining with Propidium Iodide (PI)
- Wash cells at least once with COLD PBS.
- Resuspend cells in 300 – 500 µl PI/Triton X-100 staining solution: to 10 ml of 0.
- Incubate 37°C for 15 minutes or for 30 min at 20°C.
- Transfer tubes to ice or store at 4°C PROTECTED FROM LIGHT.
How is PI used in DNA?
Propidium iodide (or PI) is a fluorescent intercalating agent that can be used to stain cells and nucleic acids. PI binds to DNA by intercalating between the bases with little or no sequence preference.
How do you dissolve PI?
Propidium iodide (PI) is supplied as a crystalline solid. A stock solution may be made by dissolving the PI in the solvent of choice. PI is soluble in organic solvents such as ethanol, DMSO, and dimethyl formamide, which should be purged with an inert gas.
What is Pi in flow cytometry?
Propidium iodide (PI) is a membrane impermeant dye that is generally excluded from viable cells. It binds to double stranded DNA by intercalating between base pairs. PI is excited at 488 nm and, with a relatively large Stokes shift, emits at a maximum wavelength of 617 nm.
Can I use PE and PI together?
PI is excited at 488 nm and, with a relatively large Stokes shift, emits at a maximum wavelength of 617 nm. Because of these spectral characteristics, PI can be used in combination with other fluorochromes excited at 488 nm such as fluorescein isothiocyanate (FITC) and phycoerythrin (PE).
What is the role of Pipi/RNase in cell cycle analysis?
PI/RNase is commonly used as a nuclear stain in fluorescent microscopy and as a DNA content determinant in cell cycle analyses by flow cytometry. Cells in G2 and M phases of the cell cycle contain twice the DNA content compared to those in G0 and G1 phases. DNA content during S phase lies between these extremes.
How to prepare DNA for flow cytometry with PI staining?
For easy setup, with PI staining of DNA content for flow cytometry we recommend our Propidium Iodide Flow Cytometry Kit, otherwise, we recommend this protocol. Harvest the cells in the appropriate manner and wash in PBS. Fix in cold 70% ethanol.
What is Pipi/RNase stain used for?
PI/RNase is commonly used as a nuclear stain in fluorescent microscopy and as a DNA content determinant in cell cycle analyses by flow cytometry. Cells in G2 and M phases of the cell cycle contain twice the DNA content compared to those in G0 and G1 phases.
How do you stain DNA with RNA?
Specific DNA staining is achieved by enzymatic removal of RNA with a ribonuclease (RNase). PI/RNase is commonly used as a nuclear stain in fluorescent microscopy and as a DNA content determinant in cell cycle analyses by flow cytometry.