In the present study, we explored a novel cellular staining protocol with acridine orange propidium iodide aopi and tested the antiproferative activity of vad against human lung cancer a549 cells and human prostate cancer pc3 cells. Discrimination of viable and nonviable cells in flow cytometry determination of dna content in flow cytometry. When a sample enters a flow cytometer, the particles are randomly. Detection of apoptotic cells using propidium iodide staining.
Dna content for cell cycle analysis of fixed cells with propidium iodide. Propidium iodide or pi is a fluorescent intercalating agent that can be used to stain cells. However, it can easily penetrate dead or damaged cells and as such is commonly used for identifying cell viability in a population or as a counterstain in multicolor fluorescent techniques. Propidium iodide viability on a plate reader bmg labtech. Propidium iodide pi and hoechst 33342 double staining in cultured c6 cells 72 hours after 400. Here, we present a highresolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. Propidium iodide pi is membrane impermeant and generally excluded from viable cells. Please read the following cell viability protocol in its entirety before beginning. I believe that methanol washes may be a way to remove the staining in the yolk, but another zebrafish expert may have more information than me. Protocol for staining dna with propidium iodide for cellcycle analysis using the bd facsarray bioanalyzer. Isolation of the side population in mycinduced tcell acute lymphoblastic leukemia in zebrafish margaret m. Analysis of nephron composition and function in the adult. The first two are based on univariate analysis of cellular dna content following cell staining with either propidium iodide pi or 4. The most commonly used dye for dna contentcell cycle analysis is propidium iodide pi.
Many debilitating diseases, including neurodegenerative diseases, involve apoptosis. Procedure for the early detection of apoptosis using annexin v staining, and optional propidium iodide pi. Distribution of ploidy levels within the plant tissues can be calculated based on the distribution of fluorescence signals. The protocol is a modified version of the standard in situ hybridization. Toescu division of medical sciences, university of birmingham 042008. Spin cells again and aspirate most of the pbs, leaving a small. 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. Livedead baclight bacterial viability and counting kit. Propidium iodide staining for dna content reagents.
We counterstain with propidium iodide pi, a nucleic acid binding dye that is excluded from the membranes of healthy cells. Verify with protocol immunologist or check the piis for each protocol found on the actg website. Analysis of bacterial cultures using the livedead baclight bacterial viability and counting kit. For these applications we recommend use of tonbo biosciences ghost dyes. Several methods have been developed for visualizing apoptotic cells in vitro or in fixed tissues, but few tools are available for visualizing apoptotic cells in live animals. Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Fluorescein diacetate fda and propidium iodide pi were used for the first time to assess viability of zebrafish ovarian follicles. Propidium iodide staining of cells to assess dna c abcam.
The method used will depend on the experiment and the information required. In this technique, cells are stained with a dye that fluoresces on dna binding, such as hoechst 33342 or propidium iodide. Analysis of cell cycle by flow cytometry 303 cytometers are becton dickinson immunocytometry systems, beckmancoulter inc. Propidium iodide is a suspected carcinogen and should be handled with care. If media is not used within 30 days, fresh lglutamine must be added before use to a final concentration of 2. Dna content for cell cycle analysis of fixed cells with.
These dyes cannot pass through intact cell membranes. Highthroughput method for dynamic measurements of cellular viability using a bmg labtech microplate reader. Analysis of apoptosis by propidium iodide staining and. Because the intact membrane of live cells excludes charged dyes such as propidium iodide pi. Evaluation of zebrafish danio rerio ovarian follicle. It is an ethidium bromide analog that emits red fluorescence upon intercalation with doublestranded dna. Novel cellular staining protocol and cellular viability. Flow cytometry basics guide 3 1 principles of the flow cytometer fluidics system one of the fundamentals of flow cytometry is the ability to measure the properties of individual particles.
Cells emitting blue fluorescence were hoechst positive while those with red fluorescence were pi positive. Staining of intracellular antigens for flow cytometry protocols. Here we describe a genetically encoded fluorescent reporter protein that labels apoptotic cells in live zebrafish embryos. Propidium iodide pi and hoechst 33342 double staining. Propidium iodide may be used in flow cytometry to evaluate cell viability when used with other dyes that stain viable cells or cells that are early in the apoptosis process. When excited by 488nm laser light, it can be detected with in the petexas red channel with a bandpass filter 61010. Measuring cell ploidy level in arabidopsis thaliana by. Since its introduction, the propidium iodide pi flow cytometric assay has been widely used for the evaluation of apoptosis in different experimental models. Described are four widely used procedures to analyze the cell cycle by flow cytometry. The best resource is probably the protocols you can find in the zebrafish book at. To study this interesting possibility, the zebrafish. The appearance of phosphatidylserine ps residues normally hidden within the plasma membrane on the surface of the cell is an early event in apoptosis, and can be used to detect and measure apoptosis.
Cell membrane integrity excludes propidium iodide from staining viable and apoptotic cells. Dna in the nuclei is stained by propidium iodide and the fluorescence emitted from the dna of each nucleus is read by using a flow cytometer. Determination of sperm concentration using flow cytometry. The aim of the present study was to develop a new viability assessment method for zebrafish ovarian follicles that is reliable, sensitive and notstage specific. A bright yellowgreen glow is produced and is strongest when enzymatic activity is greatest. Pdf propidium iodide pi uptake assay to detect apoptosis. The genomic dna extraction procedure was adapted from an established protocol gavrieli et al. 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. Pi binds to dna by intercalating between the bases with little or. Evaluation of zebrafish danio rerio ovarian follicle viability by simultaneous staining with fluorescein diacetate and propidium iodide article pdf available in cryo letters 296. Cellular dna content can be measured by flow or laserscanning cytometry with the aim of 1 revealing cell distribution within the major phases of the cell cycle, 2 estimating the frequency of apoptotic cells with fractional dna content, andor 3 disclosing the dnaploidy of the measured cell population. In this protocol, we describe a propidium iodide pi flow cytometry assay to evaluate bcell lymphomas that have undergone apoptosis in vivo. Analysis of nephron composition and function in the adult zebrafish kidney. Cellcycle analysis using the bd facsarray bioanalyzer.
Analysis of cellular dna content by flow cytometry. Zebrafish whole mount highresolution double fluorescent. Analysis of cell cycle by flow cytometry springerlink. This protocol uses ethanol to fix and permeabilize cells for staining of dna in intact cells with propidium iodide pi. A profile of the cell cycle in disaggregated zebrafish embryos or adult tissue can be obtained through dna content analysis. Staining dead cells with propidium iodide pi or 7aminoactinomycin d 7aad propidium iodide and 7aad can be used to stain dead cells so that they may be excluded from analysis in standard live cell surface staining protocols. The procedures for working with zebrafish described in this protocol were approved by the institutional animal care and use committee at the university of notre dame. Propidium iodide cell viability flow cytometry protocol. Propidium iodide pi is a popular redfluorescent nuclear and chromosome counterstain. It is commonly used in evaluation of cell viability or dna content in cell cycle analysis by flow cytometry. Propidium iodide pi uptake assay to detect apoptosis article pdf available in cold spring harbor protocols 20062 july 2006 with 2,296 reads how we measure reads. The result is red fluorescence surrounding each cell in the root. The first protocol for cell cycle analysis using propidium iodide staining was presented in 1975 by awtar krishan from harvard medical school and is still widely cited today.
Introduction to flow cytometry flow cytometry is a popular laserbased technology. Since propidium iodide is not permeant to live cells, it is also commonly used to detect dead cells in a population. The pi intercalates into the major groove of doublestranded dna producing a. Annexin v staining assay protocol for apoptosis abcam. Propidium iodide pi is a fluorescent dye that binds to dna. Cell nuclei are isolated by filtering tissue homogenates from chopped plant tissues. Pi is excited at 488535 nm with an emission maximum of. Technical data sheet propidium iodide tonbo biosciences. Pi binds to dna by intercalating between the bases with little or no sequence preference. Regain access you can regain access to a recent pay per article purchase if your access period has not yet expired. It can be used to stain whole cells or isolated nuclei. Fluorescein diacetate fda hydrolysis assays can be used to measure the enzyme activity of microbes in a sample. Propidium iodide pi is a fluorescent dye which intercalates between bases and stains both dna and rna. In this application note we show that the propidium iodide assay is a very useful tool for monitoring neuronal viability.
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