Pi binds to dna by intercalating between the bases with little or no sequence preference. Cell cycle analysis by flow cytometry uses a dna binding dye, such as propidium iodide pi, 7 aminoactinomycin d 7aad or 4, 6diamidino2phenylindole dapi, to determine the cell cycle state. It is commonly used in evaluation of cell viability or dna content in cell cycle analysis by flow cytometry. This propidium iodide solution includes dnasefree rnase a and a. Propidium iodide pi is a popular redfluorescent nuclear and chromosome counterstain. Designed for ease of use in cell sorting and flow cytometry applications, the readidrop propidium iodide cell viability assay removes the traditional manual weighing, pipetting, and dilution steps. The cell cycle is the process by which eukaryotic cells duplicate and divide. 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 dnacontaining organelles. Application note this application note describes a method for using the bd facsarray bioanalyzer to analyze cells stained for cellcycle analysis using propidium iodide and an additional intranuclear stain for multiparameter analysis. The most commonly used dye for dna content cell cycle analysis is propidium iodide pi. Add this solution to 1 ml of a cell suspension containing 10,000,000 cells. Flow cytometric analysis with propidium iodide pi is widely employed in identifying the accumulation of cells in different phases of cell cycle based on their dna content 14.
Pdf analysis of cell cycle by flow cytometry methods. Then apply this gate to the scatter plot and gate out obvious debris. The first is based on the simultaneous analysis of proliferation specific marker ki67 and cellular dna content, which discriminates restingquiescent cell populations g0 cell and quantifies cell cycle distribution g1, s or g2m, respectively. Before analysis, the cells are usually permeabilised and treated with a fluorescent dye that stains dna quantitatively, such as propidium iodide. Cellcycle analysis using the bd facsarray bioanalyzer. Cell cycle analysis in fixed cells thermo fisher scientific sg. Propidium iodide or pi is a fluorescent intercalating agent that can be used to stain cells and nucleic acids. Flow cytometry cell cycle analysis using propidium iodide dna staining. Resuspend cell pellet in 500 l nucleic acid staining solution. Propidium iodide pi is a redfluorescent cell viability dye which is excluded from live cells as it is impermeable to the cell membrane. Resuspend cells in 300 500 l pitriton x100 staining solution. A stock solution of pi, made by dissolving 1 mg pi in 2 ml water, can be stored several months at 0 to 4c. Cell cycle analysis by quantitation of dna content was one of the earliest applications of flow cytometry. Measuring drug effect on cell cycle profile using the.
Propidium iodide pi is widely used for staining and evaluation of cell death and apoptosis or for determination of dna content in cell cycle analysis. Combine the gates and apply to the pi histogram plot. The cell cycle profile of a sample can be determined by staining the dna with a fluorescent. Fxcycle pirnase staining solution is used for flow cyotmetric analysis of dna content in fixed cells. Cell cycle analysis by quantitation of dna content was one of the earliest applications of flow. Dna content for cell cycle analysis of fixed cells with propidium iodide. The countstar system combines the image cytometer and cell counter into a single benchtop instrument. Compared to this analysis method, the cell cycle assay solution deep red has good membrane permeability and uses the dye with high specificity for dna, which makes it possible to analyze the cell cycle just by adding the reagent to a cell suspension. Add 1 ml of propidium iodide staining solution to cell pellet and mix well. Complex hypotonic solution the following protocol is for. Propidium iodide solution apoptosis and cell viability.
Propidium iodide staining of cells to assess dna c abcam. For cell cycle analysis even you can store sample at 20c for several days. Dna content for cell cycle analysis of fixed cells with propidium. Propidium iodide pi is generally used for cell cycle analysis using flow cytometry. Propidium iodide staining of cells for cell cycle analysis protocol. The propidium iodide dye exclusion assay is performed in a krebsringerhepes buffer krh. In the absence of cells, propidium iodide exhibits an excitation maximum near 500 nm and an emission maximum near 625 nm. The dye intercalates dna and rna of the dead cells to give red. Rapid flow cytofluorometric analysis of mammalian cell cycle.
Rapid flow cytofluorometric analysis of mammalian cell. And if someone kindly tell me how you prepare propidium iodide solution. Since propidium iodide is not permeant to live cells, it is also commonly used to detect dead cells in a population. Cancer cell analysis for research propidium iodide. Propidium iodide pi bluegreen lasers, also stains rna. Combining dna content analysis with cell cycle checkpoint markers for assessing additional cell cycle compartments beckman coulter life sciences is taking actions in the best interests of our associates. Cell cycle analysis using pi cell cycle analysis is used to determine the proportion of cells in each stage of the cell cycle for a given cell population based on variations in dna content.
Cell cycle analysis by propidium iodide pi staining. By measuring the dna content of individual cells, we obtain information about their ploidy seesection 6. For cell cycle analysis, please see our propidium iodide cell cycle staining protocol. Analysis of apoptosis by propidium iodide staining and. Fl3 propidium iodide, and set a division line for positivenegative on each color it will automatically set the division for 99% negative on your control cells. The relationship between the dna histogram and the cell cycle. This applicationdriven, compact, and automated cell imaging system provides an allinone solution for cancer cell research, including cell counting, viability aopi, trypan blue, apoptosis annexin vfitcpi, cell cycle pi, and gfprfp transfection. This allows the user to analyze cells for gfp content versus cell cycle without loosing significant amounts of gfp or use other channels for antibody fluorochrome detection with cell cycle analysis without the need for a uv laser line. There are two ways to quantitate the percentage of cells in each cell cycle phase.
Propidium iodide is not membranepermeable, making it useful to differentiate necrotic, apoptotic and healthy cells based on membrane integrity. Cell cycle analysis is a very common flow cytometry application. Add 50 ul of rnase a stock solution final concentration 0. Analysis of apoptosis by propidium iodide staining and flow. Propidium iodide is a fluorescent intercalating agent used for staining. One method to test cell viability is using dye exclusion. If the research requires more stringent analysis the flow cytometry core facility has deconvolution software that allows the investigator to calculate the area under the curve for g 1, s phase and g 2m. Quantitation of cell cycle phases by combined propidium iodide and brdu staining pibrdu. Propidium iodide pi is a membrane impermeant dye that is generally excluded from viable cells. Resuspend at 2 x 10 6 cells in 1 ml ice cold buffer. Also provides cell cycle analysis, neural net clustering, batch mode support, composite graph generation from.
Commonly used dna dyes used for flow cytometric cell cycle analysis include propidium iodide pi, 4,6diamidino2phenylindole dihydrocloride dapi. Propidium iodide pi is a readytouse solution for the exclusion of nonviable cells in flow cytometric analysis. By using a dnaspecific stain, one can determine a dna profile e. Cell cycle analysis with propidium iodide pi staining.
Propidium iodide pi is a fluorescent dye that binds to dna. Analyzing cell cycle using dyes designed for the violet, blue, or red lasers increases the. This is available with some flow cytometry software and is more objective than setting. Dna content for cell cycle analysis of fixed cells with. The software used to deconvolute the dna content frequency histograms, to estimate the proportions of cells in the respective phases of the cycle, is available. Propidium iodide cell viability flow cytometry protocol. View a selection guide for all products related to cell cycle analysis of fixed and live cells in flow cytometry. Compared to this analysis method, the cell cycle assay solution deep red has good membrane permeability and uses the dye with high specificity for dna, which makes it possible to analyze the cell cycle just by adding the reagent to a cell. It is commonly used in evaluation of cell viability or dna content in cell cycle analysis. The prodidium iodide pi staining solution may be used to assess plasma membrane pm integrity in annexin v apoptosis assays. Propidium iodide pi bluegreen lasers, also stains rna dapi uvviolet lasers. Chagas disease is a neglected tropical disease caused by the protozoan parasite trypanosoma cruzi which affects 68 million people, mostly in latin am.
Propidium iodide solution life science research biorad. Adapted from current protocols in cytometry this protocol uses ethanol to fix and permeabilize cells for staining of dna in intact cells with propidium iodide pi. For analysis, first gate on the single cell population using pulse width vs. Live hela cells stained with cellsimple propidium iodide pi rnase staining solution kit were analyzed on the cellsimple cell analyzer using the cell cycle app. Propidium iodide pi, a popular redfluorescent stain, binds to dna by intercalating between the bases with little or no sequence preference and with a stoichiometry of one dye per 45 base pairs of dna. This analysis distinguishes cells in different phases of cell cycle and is often used to. Cell cycle analysis by propidium iodide pi staining adherent cells. Staining is with a 50ugml solution of pi containing a 0. Note that cells with 2n and 4n dna content are centered over the 200 and 400 marks on the xaxis scale for propidium iodide staining intensity. Propidium iodide pi is a fluorescent dye which intercalates between bases and stains both dna and rna. Read how propidium iodide staining can be used to assess cell cycle state using flow cytometry. The pi intercalates into the major groove of doublestranded dna producing a. Pi can be used in fl3 for inviability exclusion, but should be analyzed in fl2 when used as. Protocol propidium iodide cell cycle staining protocol.
Application note this application note describes a method for using the bd facsarray bioanalyzer to analyze cells stained for cell cycle analysis using propidium iodide and an additional intranuclear stain for multiparameter analysis. Propidium iodide pi is a nuclear staining dye that is frequently used to measure cell cycle. Propidium iodide pi is a redfluorescent cell viability dye which is excluded from live cells with intact membranes, but penetrates dead or damaged cells and binds to dna and rna by intercalating. Store at 4c propidium iodide pi rnase staining solution kit. Jan 21, 2012 quantitation of cell cycle phases by combined propidium iodide and brdu staining pibrdu. Flow cytometry histogram left further analyzed using modfit lt verity software house, topsham, me to obtain fitting for cell cycle fraction data right. The suggested use of this solution for viability staining is 10 l per million cells in 0. The dna of mammalian, yeast, plant or bacterial cells can be stained by a variety of dna binding dyes. The cell cycle consists of two specific and distinct phases. Cell cycle analysis with propidium iodide pi staining cell line. Cell cycle analysis by dna content propidium iodide page content. Gates were positioned to place the cell cycle cluster into the highlighted region in the upper right region of the plot. Dna cell cycle analysis with pi cornell university college. Propidium iodide pi is a nuclear staining dye that is frequently used to measure.
Labeling and staining cells with brdu, combined with total dna staining by propidium iodide and analysis by flow cytometry 1 offers the most accurate measure of cells in the various stages of the cell cycle. Pipet the 1 ml cell suspension into 3 ml 4% paraformaldehyde and fix for 15 min at r. The results are amazing and it is possible to get cvs under 10% with most cell lines. The propidium iodide should be read on the appropriate channel in the linear scale. Dna analysis is, after immunofluorescence, the second most important application of flow cytometry. Flow cytometric analysis of cell cycle with propidium iodide dna staining. It does not cross the pm of cells that are viable or in the early stages of apoptosis because they maintain pm integrity. The staining procedure takes less than 1 hour of total processing time and cells fixed in ethanol are. This propidium iodide solution includes dnasefree rnase a and a permeablization reagent.
Cell cycle analysis by dna content measurement is a method that most frequently employs flow cytometry to distinguish cells in different phases of the cell cycle. Propidium iodide staining of cells for cell cycle analysis. Cell cycle analysis software cell cycle analysis of research samples can be adequately done by the straight linear gating as illustrated above. Propidium iodide pi is a redfluorescent cell viability dye which is excluded from live cells with intact membranes, but penetrates dead or damaged cells and binds to dna and rna by intercalating between the bases. Cells exhibiting fluorescence above 630 nm should be excluded from further analysis. Each cell is stained with a fluorescent dye that intercalates with dna. In this unit, we describe two protocols for analyzing cell cycle status using flow cytometry. Note that cells with 2n and 4n dna content are centered over the 200 and 400 marks on the xaxis scale for propidium iodide.
The following protocol is for dna staining with propidium iodide for cell cycle analysis complex hypotonic solution. Analysis of cell cycle by flow cytometry 303 cytometers are becton dickinson immunocytometry systems, beckmancoulter inc. Cell cycle with propidium iodide nexcelom bioscience. It can be used to stain whole cells or isolated nuclei. Catalog of free flow cytometry software purdue university. Can anyone recommend a good free facs analysing software. Store at 4c propidium iodide pirnase staining solution rev. Store samples at 4 c until analyzed by flow cytometry. Sigmaaldrich offers a number of propidium iodide solution products. The adam mc can work with a dizzying range of suspension cell. A this panel displays three dimensional flow cytometry of propidium iodide and brdu stained cells. This analysis distinguishes cells in different phases of cell cycle. Flow cytometry histogram left further analyzed using modfit lt verity software house, topsham, me to obtain fitting for cell cycle.
Resuspend cell pellet in 500 ul nucleic acid staining solution. The propidium iodide solution is suitable for the exclusion of dead cells from flow cytometric analysis. Analysis of cell cycle position in mammalian cells protocol. Pi can be used in fl3 for inviability exclusion, but should be analyzed. Live cells have membranes that are still intact and exclude a variety of dyes that easily penetrate the damaged, permeable membranes of nonviable cells.
Propidium iodide staining of dna is the classic means of cell cycle analysis. When excited by 488nm laser light, it can be detected with in the petexas red channel with a bandpass filter 61010. Cell cycle phasespecific analysis of cell viability using hoechst 33342 and propidium iodide after ethanol preservation. Pi staining solutions provided are a reasonable starting point for concentrations of fluorochrome, however, this will vary with cell. Pi binds to double stranded dna by intercalating between basepairs, but is excluded from cells with intact plasma membranes. The software used to deconvolute the dna content frequency histograms, to estimate the proportions of cells in the respective phases of the cycle. Specific dna staining is achieved by enzymatic removal of rna with a ribonuclease rnase. Immediately before flow cytometric analysis, prepare a 0. View a selection guide for all products related to cell cycle analysis. What is the best protocol for preparing sample for facs cell cycle.
1336 1541 1328 941 34 949 568 1466 735 1247 1588 764 825 240 1414 1010 991 1624 402 929 1376 1380 729 956 727 172 457 781 1199 37 460 66 863 180 265 715 405