How does propidium iodide work in flow cytometry?
Flow Cytometry Cell Viability Overview Flow cytometry is a quick and reliable method to quantify viable cells. 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.
What is the concentration of propidium iodide for FACS?
1 μg/mL
It is recommended to use Propidium Iodide Solution at a final concentration of 1 μg/mL.
What does propidium iodide do to cells?
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.
What is propidium iodide assay?
A propidium iodide fluorescence assay (PIA) was developed to characterize the in vitro growth of human tumor cell lines as well as to test the cytotoxic activity of standard compounds. Propidium iodide (PI) was used as a dye which penetrates only damaged cellular membranes.
How do you make propidium iodide 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 determine cell cycle?
The most common method for assessing the cell cycle is to use flow cytometry to measure cellular DNA content. During this process, a fluorescent dye that binds to DNA is incubated with a single cell suspension of permeabilized or fixed cells.
What is propidium iodide staining?
Propidium iodide (or PI) is a fluorescent intercalating agent that can be used to stain cells and nucleic acids. Propidium iodide is used as a DNA stain in flow cytometry to evaluate cell viability or DNA content in cell cycle analysis, or in microscopy to visualize the nucleus and other DNA-containing organelles.
Is propidium iodide soluble in water?
Solubility : Soluble in water (1 mg/ml), DMSO, and methanol (3 mg/ml). Melting Point : 220-225° C (dec.)
How is propidium iodide used in flow cytometry?
Flow cytometry cell cycle analysis using propidium iodide DNA staining. Introduction to flow cytometric analysis Cell cycle analysis by quantitation of DNA content was one of the earliest applications of flow cytometry. The DNA of mammalian, yeast, plant or bacterial cells can be stained by a variety of DNA binding dyes.
How long does it take to fix propidium iodide?
Add dropwise to the cell pellet while vortexing. This should ensure fixation of all cells and minimize clumping. Fix for at least 30 minutes at 4°C. Specimens can be left at this stage for several weeks at -20°C.
How is propidium iodide excluded from a viable cell?
Live cells have membranes that are still intact and exclude a variety of dyes that easily penetrate the damaged, permeable membranes of non-viable cells. Propidium iodide (PI) is a membrane impermeant dye that is generally excluded from viable cells.
How to fix propidium iodide cells in PBS?
Harvest cells in the appropriate manner and wash in PBS. Fix in cold 70% ethanol (do not make this with PBS as it can cause protein precipitation during fixation). Add dropwise to the cell pellet while vortexing. This should ensure fixation of all cells and minimize clumping.
