What is a PI solution?
Propidium iodide (or PI) is a fluorescent intercalating agent that can be used to stain cells and nucleic acids. When in an aqueous solution, PI has a fluorescent excitation maximum of 493 nm (blue-green), and an emission maximum of 636 nm (red).
How do you make a PI solution?
PI Stock Solution (1mg/ml or 1.5 mM): To prepare, add 1 mg PI (Propidium Iodide) to 1 ml distilled water. Store stock solution at 4 ºC (or aliquot and store at -20 ºC), protecting from light. When handled properly, solutions are stable for many years.
How do you use PI stain?
Propidium Iodide (PI) Staining
- Protocol 1. Step 1: Harvest and count cells. Step 2: Centrifuge and remove supernatant.
- Protocol 2 Fixation. Transfer the medium with floating cells (4ml from 60mm dish) into 15 ml centrifuge tube.
- Staining. Briefly vortex then spin fixed cells down at 3000 rpm for 5 min.
Is propidium iodide water soluble?
Solubility : Soluble in water (1 mg/ml), DMSO, and methanol (3 mg/ml). Melting Point : 220-225° C (dec.)
Does PI stain apoptotic cells?
The ability of PI to enter a cell is dependent upon the permeability of the membrane; PI does not stain live or early apoptotic cells due to the presence of an intact plasma membrane 1,2,6.
What does propidium iodide detect?
Propidium iodide (PI) is a popular red-fluorescent nuclear and chromosome counterstain. 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.
How do you dilute pi?
1) Weigh out PI and place in 15ml conical tube. 2) Dilute PI to 1 mg/ml with distilled water. 3) Cover tube with foil and store refrigerated.
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.
Can I fix cells after PI staining?
Propidium Iodide is a charged substance; i.e. the positively charged propidium ion does not enter living cells. If the aim is to stain all cells, then they must be fixed in ethanol before staining.
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).
How do you reconstitute propidium iodide?
Reconstitute propidium iodide powder with 3.0 mL of PBS to a final concentration of 0.33 mg/mL. The reconstituted stock solution should be stored at -20°C and is stable for approximately 6 months.
What is the difference between FDA and PI staining?
FDA is taken up by cells which convert the non-fluorescent FDA into the green fluorescent metabolite fluorescein. The measured signal serves as indicator for viable cells, as the conversion is esterase dependent. In contrast, the nuclei staining dye PI cannot pass through a viable cell membrane.
Does Pipi stain crosslink nucleic acids?
PI and other nucleic acid stains do not inherently bind covalently to nucleic acids and these fixatives do not crosslink the dyes to nucleic acids. The one fixable nucleic acid stain is Ethidium Monoazide Bromide (EMA), Cat no. E1374); it covalently binds to nucleic acids upon activation by exposure to light.
How do you make a stock solution from solid Pi?
To make a stock solution from the solid form, dissolve PI (MW = 668.4) in deionized water (dH2O) at 1 mg/mL (1.5 mM) and store at 2–6°C, protected from light. When stored properly, solutions are stable for at least six months.
What is the staining protocol for adherent cells?
The staining protocol is applicable to adherent cells, single cells embedded in extracellular matrix and 3D cell clusters, for example multicellular spheroids. 2 Principle Live/dead staining can be performed with FDA and PI.