Cell cycle analysis by propidium iodide pi staining. However, different staining protocols may be necessary for some experiments. 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. Propidium iodide solution apoptosis and cell viability.
The above experiment was done using pi propidium iodide which stains dna and rna by intercolating between the bases. It is our preferred method because it combines the detection of active dna synthesis, through antibody based staining of brdu, with total. Propidium iodide staining of cells for cell cycle analysis. Cell apoptosis assay is a type used for determining the apoptosis percentage of cells by annexin v. The cell cycle profile of a sample can be determined by staining the dna with a fluorescent dye and measuring its intensity. After collecting cell sample control as well as treated, i fix it with 70% ethanol overnight. Doublet discrimination when measuring cell cycle by flow cytometry, there is always a chance that doublets will complicate and skew your results. Pipet the 1 ml cell suspension into 3 ml 4% paraformaldehyde and fix for 15 min at r. Lastly, for in vivo cell cycle analysis, brdu can be injected in rodents and the target cell population can be isolated for cell cycle.
Guava cell cycle software includes markers that can be set for quick assessment of g0g1, s, and g2m phase cell cycle percentages during data collection for rapid screening of compounds. Assaying cell cycle status using flow cytometry ncbi nih. 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. Analysis of cell cycle by flow cytometry 303 cytometers are becton dickinson immunocytometry systems, beckmancoulter inc. The cell cycle is the process by which eukaryotic cells duplicate and divide. Pdf analysis of cell cycle by flow cytometry methods. Dna content for cell cycle analysis of fixed cells with propidium. This system uses bd facsuite software for all functions from. The diverse software provides several mathematical models for fitting the dna histogram. Dapi and hoechst 33342 can also be used to determine the cell cycle of cells with a violet 405nm laser diode giving cvs of the g1 peak of 6%. Propidium iodide pi is a nuclear staining dye that is frequently applied in measuring cell cycle. Propidium iodide pi is used to stain the dna of isolated nuclei in a.
Cell cycle analysis with propidium iodide pi staining. One method to test cell viability is using dye exclusion. Cell cycle analysis with propidium iodide pi staining cell line. Bd facsuite software provides an easytouse yet flexible interface for data acquisition and analysis with the bd facslyric system. The relationship between the dna histogram and the cell cycle. 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. The cell cycle profile of a sample can be determined by staining the dna with a fluorescent. Cellcycle analysis an overview sciencedirect topics. By using a dnaspecific stain, one can determine a dna profile e. Since the amount of bound fluorescent dye is directly proportional to the amount of dna present within a cell. In the absence of cells, propidium iodide exhibits an excitation maximum near 500 nm and an emission maximum near 625 nm. Propidium iodide staining of cells for cell cycle analysis protocol this method provides a general procedure for dna staining for cell cycle analysis using propidium iodide pi.
Propidium iodide pi is the most commonly used fluorochrome, owing to its optimal linear dnabinding capacity in a variety of different cell types. Cellcycle analysis using the bd facsarray bioanalyzer. Flow cytometric analysis of cell cycle with propidium iodide dna staining. Propidium iodide pi is used to stain the dna of isolated nuclei in a stoichiometric. The protocol determining cellular dna content in cell cycle analysis cell apoptosis. The gap1 g1 phase of an eukaryotic cell is defined as having 2c dna. The most commonly used dye for dna content cell cycle analysis is propidium iodide pi. Below are some examples of cell cycle data acquired using a bd lsr ii flow cytometer and analysed with fcs express software. The cell cycle aka, cell division cycle consists of a series of processes involved in the growth, replication, and division of. Certain flow cytometry data analysis softwares are equipped with automated gating options for pistained cells. This singleaddition reagent is composed of propidium iodide. Propidium iodide pi is a nuclear staining dye that is frequently used to measure. Dna cell cycle analysis with pi cornell university college. This singleaddition reagent is composed of propidium iodide pi, rnase a, and triton x100.
Page 3 when not in the process of preparing for cell division, most of the cells in our body are not, cells remain in the g1 portion of the cell cycle. Cell cycle analysis involves propidium iodide staining of the cells population and shows how many of the cells are found in different cell cycle phases 180. Based on an established method of whole cell staining with propidium iodide pi, the guava cell cycle assay is easy to perform. The cellcycle aka, cell division cycle consists of a series of processes involved. Cell cycle analysis by propidium iodide pi staining adherent cells. Catalog of free flow cytometry software purdue university. The g1 phase is thus numerically the most predominant phase. Commonly used dna dyes used for flow cytometric cell cycle analysis include propidium. This protocol uses ethanol to fix and permeabilize cells for staining of dna in intact cells with propidium iodide pi. Appropriate controls should always be carried out, for. Dapi and hoechst 33342 can also be used to determine the cell cycle of cells with a violet 405nm laser diode giving cvs of the g1 peak of 6% as opposed to propidium iodide solution is suitable for the exclusion of dead cells from flow cytometric analysis. Dna content cell cycle analysis using flow cytometry youtube.
Fluorescencebased cell cycle analysispart 1 nexcelom. Cycle analyzer ploidy determinations are no longer bound to the peak order. Cell cycle analysis software cell cycle analysis of research samples can be adequately done by the straight linear gating as illustrated above. All normal analysis capabilities exist in the free version. However, a subtraction of the background is required in order to remove events due. Multiparametric cell cycle and immunophenotypic analysis. Time voltageintensity area for each cell or debris particle, the electronics measure the area and height values. The pi intercalates into the major groove of doublestranded dna producing a. 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. Cells may form a diffuse ringshaped pellet, so centrifuge longer e.
This produces a histogram of the propidium iodide concentration for each cell as it passes through the laser. Each cell is stained with a fluorescent dye that intercalates with dna. The tali cell cycle kit includes a readytouse reagent that, when used with the tali imagebased cytometer, provides a convenient and accurate determination of the percentage of cells in each phase of the cell cycle. Cell cycle and dna content analysis using the bd cycletest assay on. Propidium iodide cell viability flow cytometry protocol. I want to analyze cell cycle apoptosis specifically using facs with propidium iodide staining. Width is calculated by dividing area by height x 64k. Also provides cell cycle analysis, neural net clustering, batch mode support, composite graph generation from multiple files, up to 10 rectangular or irregular regions of interest for color highlighting between parameters or editing, and several forms of hardcopy output ps, hpgl, pcl, gif, pict. 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. In cycling cells, there will be two strong peaks connected in the center like the letter u.
The countstar system combines the image cytometer and cell counter into a single benchtop instrument. The propidium iodide solution is suitable for the exclusion of dead cells from flow cytometric analysis. Cell cycle assays for flow cytometry thermo fisher. I am analyzing cell cycle data from flow cytometry using pi as a single dye, and i encountered several. The sophisticated peak detection system identifies ploidy patterns and dynamically creates models to match. This is a prerequisite for other dyes commonly used for measurement of cell cycle phases, such as propidium iodide. Using fluorescence microscopy and image analysis, the nc3000 system automates dna content quantification and hence, measurements of cell cycle stages. Cell cycle analysis software, for example, modfit lt verity software house back to top. The application is designed for userdefined assays and collaboration through the assay portability feature, and provides a simple, reproducible and transferrable setup. In addition to surface immunophenotyping and cytoplasmic characterization, flow cytometry is also used in cell cycle analysis. Based on an established method of wholecell staining with propidium iodide pi, the guava cell cycle assay is easy to perform. The cell cycle consists of two specific and distinct phases.
Introduction one of the most common and popular methods for cell cycle detection is the use of fluorescencebased dyes. The premise with these dyes is that they are stoichiometric i. Nov 09, 2017 time voltageintensity area for each cell or debris particle, the electronics measure the area and height values. Cell cycle analysis by quantitation of dna content was one of the earliest applications of flow cytometry. Dna content for cell cycle analysis of fixed cells with. Autodebris, autoaggregate, and autolinearity make analysis robust and consistent. Software for cell cycle analysis diverse manufacturers such as becton dickinson have developed software cellfit for cell cycle analysis. Before analysis, the cells are usually permeabilised and treated with a fluorescent dye that stains dna quantitatively, such as propidium iodide pi or 4,6diamidino2phenylindole dapi. The most straightforward method for cell cycle analysis is to fix the cells with ethanol, treat with rnase, and stain with pi. Results demonstrated that the breast cancer cells progressed through cell cycle at a slower rate compared to the colorectal cell lines, given the higher percentage of breast cancer cells in the g1 over time. Protocol propidium iodide cell cycle staining protocol biolegend. Staining is with a 50ugml solution of pi containing a 0.
Resuspend cells in 300 500 l pitriton x100 staining solution. Propidium iodide staining of cells to assess dna c abcam. The most commonly used dye for dna contentcell cycle analysis is propidium iodide pi. Countstar rigel s6 advanced image cytometer cell analyzer.
These are guidelines only and the incubation times may need to be adjusted for different cell types. Cell cycle analysis using flow cytometry one can determine what percentage of a cell population is in which part of the cell cycle. Dna content for cell cycle analysis of fixed cells with propidium iodide. Cell cycle analysis by dna content protocols flow cytometry. The propidium iodide dye exclusion assay is performed in a krebsringerhepes buffer krh. Flow cytometry histogram left further analyzed using modfit lt verity software house, topsham, me to obtain fitting for cell cycle fraction data right.
I recommend modfit software for cell cycle analysis. Cyflogic a free flow cytometry data analysis software. This information can be used to, for example, monitor the effect of an anticancer treatment. Propidium iodide staining of dna is the classic means of cell cycle analysis. Propidium iodide pi bluegreen lasers, also stains rna.
Cell cycle, flow cytometry, ki67, propidium iodide. Jan 21, 2012 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. Dna measurement and cell cycle analysis by flow cytometry. Flow cytometry cell cycle analysis using propidium iodide dna staining. The above experiment was done using pipropidium iodide which stains dna and rna by intercolating between the bases. 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. The staining procedure takes less than 1 hour of total processing time and cells fixed in ethanol are. 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. There are a number of fluorescentbased dyes that are capable of binding to double stranded dna upon cell fixation. Propidium iodide pi is a membrane impermeant dye that is generally excluded from viable cells. This method provides a general procedure for dna staining for cell cycle analysis using propidium iodide pi. By measuring the dna content of individual cells, we obtain information about their ploidy seesection 6. Flow cytometric analysis of ki67 and dna content for analyzing cell.
Plus reagent kit, the bd cycletest assay in bd facsuite software provides. This is available with some flow cytometry software and is more objective than setting. Tf1 erythroblast cells were alcoholfixed overnight, washed, and then suspended in 0. The first peak on the left represents the g1phase of the cell cycle while the second peak on the right represents g2phase of the cell cycle. A stock solution of pi, made by dissolving 1 mg pi in 2 ml water, can be stored several months at 0 to 4c. Hovewer, if you buy a licence for your research group company, you will get the number of extremely nice features, such as cell cycle analysis. To quantify cell cycle distribution of proliferating cells precisely, fitting software such as.
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 of a cell population. It can be used to stain whole cells or isolated nuclei. Dna analysis is, after immunofluorescence, the second most important application of flow cytometry. What is the recommended practice for analyzing flow cytometry cell.