Chapter 11 dapi staining of fixed cells for highresolution flow cytometry of nuclear dna friedrich otto fachklinik homheide d4400 munster federal republic of germany i. Life technologies 29851 willow creek rd eugene, oregon 97402 usa. This time because i have different fluorochromes i cant use pi staining so i will use dapi. What is the right dapi concentration to use in a flow. Analysis of cell cycle by flow cytometry 303 cytometers are becton dickinson immunocytometry systems, beckmancoulter inc. Dapi staining has also been shown to be a sensitive and specific detection method for mycoplasma. Dapi staining for cell cycle analysis flow cytometry embl. Labeling nuclear dna using dapi cold spring harbor protocols. Dapi binds to dna to form a stable complex in the minor groove of the a. After staining with dapi, detectwith fluorescence microscope or flow cytometry. Chapter 11 dapi staining of fixed cells for highresolution flow. Introduction the use of dapi 4, 6diamidino2phenylindole for flow cytometry fcm was proposed by gohde etal. Dapi protocol for fluorescence imaging thermo fisher. Dapi nucleic acid stain 2 before you begin materials required but not provided for fluorescence microscopy pbs optional.
A flowcytometrybased method to simplify the analysis and. Furthermore, we recently implemented this approach to quantify how another key hr factor, ctip, accumulates on chromatin upon dnadamage induction 21. Alternatively, dilute dapi stock solution to a concentration between 10. It is often used to determine cell cycle progress, i think, by histology. Facs using dapi for apoptosis analysis flow cytometry. Dapi staining is usually used in cell death detection. Cell cycle determination using dapi pfa fixation dapi is commonly used in cell cycle analyses since preferentially binds dsdna when cells are permeabilized allowing it to saturate the nucleic acids.
For easy setup, with pi staining of dna content for flow cytometry we recommend our propidium iodide flow cytometry kit, otherwise, we recommend this protocol. Whole blood samples may be used undiluted unless the cell count is high, e. Ester is a suitable functional group for staining viable cells because it can pass. Dapi staining solution ab228549 is a fluorescent stain for labeling dna in fluorescence microscopy. Fluorescence spectral characteristics the excitation maximum for dapi bound to dsdna is 358 nm, and the emission maximum is 461 nm. Technical data sheet bioimaging certified rea gent dapi solution. Propidium iodide cell viability flow cytometry protocol. Titration is the process of identifying the correct concentration of antibody to use for a given assay. Technical data sheet bioimaging certified rea gent dapi. Propidium iodide stock solution 50 gml ribonuclease i stock 100 gml method. Dapi has poor solubility in water, so sonicate as necessary to dissolve. The cells can be run on the flow cytometer immediately or kept on ice in the dark for later in the day. Dilute dapi stock solution to a concentration between 1.
Cell cycle determination using dapi alcohol fixation method. Propidium iodide is a suspected carcinogen and should be handled with care. I need to check cell viability in a flow cytometry assay, and i always use pi. Also used to ensure the antibody performs within acceptable. Dapi staining of fixed cells for highresolution flow cytometry of nuclear dna. Dapi%staining%for%cell%cycle%analysis% flow cytometry. Laser excitation of 60 mwat 355 nm and a 45050 nm band pass filter was used for detection of dapi. I need help, where could i find a good protocol that uses dapi for the study of apoptosis by. Assaying cell cycle status using flow cytometry unit 28. Dapi is predominantly impermeant to live cells, allowing it to be used as a.
Both dapi and dapi dilactate work well in these protocols. Flow cytometry was done using a becton dickinson lsrii. Gfp fluorescence was detected using laser excitation of 20 mwat 488 nm and a 52550 band pass filter. However, the use of dapi in facs would require a much different protocol, i guess fixation ect. Pdf the potential of flow cytometry in the study of unpigmented. Comprehensive analysis of human pancreatic islets using flow. The method used will depend on the experiment and the information required. Loss of membrane integrity is an indicator of cell death in flow cytometric analysis. Cell cycle determination using dapi alcohol fixation method dapi is commonly used in cell cycle analyses since it preferentially binds dsdna, when cells are permeabilized, allowing it to intercalate with and saturate the nucleic acids. Dapi ready made solution for nucleic acid staining, 1 mgml.
The excitation maximum for dapi bound to dsdna is 358 nm, and the emission maximum is 461 nm. When pi was employed as the original article cytometry part a 89a. If analysis must be delayed, then the stained cells can be fixed with buffered paraformaldehyde for example, bd cytofix buffer, see the product tds for a detailed protocol for 30 minutes at 4c, washed, resuspended in stain buffer, and then stored at 4c protected from light. Sederstrom2 1 department of molecular and cellular biology, baylor college of medicine, houston, texas 2 cytometry and cell sorting core, baylor college of medicine, houston, texas. Chapter 11 dapi staining of fixed cells for highresolution. We expect that this staining and sorting protocol may greatly facilitate and. Add 2 ml of deionized water dih 2 o or dimethylformamide dmf to the entire contents of the dapi vial to make a 14. The following is a general protocol for preparing assay solutions. Preparation of cells for flow cytometry for further information. It may be used for photofootprinting of dna, to detect annealed probes in blotting applications by specifically visualizing the doublestranded complex, and to study the changes in dna and analyze dna content during apoptosis using flow cytometry. This retains the target protein in the original cellular location.
Triton, digitonin and saponin are examples of permeabilization reagents which act by disrupting the cellular membrane. Since dapi passes through an intact cell membrane, it can be used to stain live cells and fix. Mario roederers website fluorochrome conjugation to antibody. The following protocols can be modified for tissue staining or for staining unfixed cells or tissues.
The dye binds to the minor groove of dsdna with approximately 20fold fluorescence enhancement, with higher affinity for at rich regions. Carver college of medicine flow cytometry facility 431 newton road 48 eckstein medical research building. Examine immediately using appropriate excitation wavelength. Dapi will also bind to dsrna, but gives emits at a longer wavelength near 500 nm. Nonspecific antibody binding is best eliminated by optimizing the amount. The becton dickinson flow cytometers do not calculate absolute cell counts total number of cells per sample. 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. Two color flow cytometric analysis of jurkat cell viability. Titration and antibodies the basics flow cytometry staining protocol background. Flow cytometry intracellular staining protocol fix cells before intracellular staining to fixing and permeabilization ensure stability of soluble antigens or antigens with a short halflife see the special recommendations below for exceptions. In general you would want the cells at a concentration of 2 x 10 6 ml of dapi. In most cases the cells will be spun down and the supernatant removed before adding dapi.
Adjust the cell suspension to a concentration of 1 x 10 7 cellsml with cold 4 o c pbsbsa buffer. Dapi can also serve to fluorescently label cells for analysis in multicolor flow cytometry experiments. Dapi may be used in flow cytometry systems utilizing uv excitation sources. Since 1979, the flow cytometry facility has provided university of iowa investigators with stateoftheart, laserbased instrumentation for basic cell research. Please read the following cell viability protocol in its entirety before beginning. Also used to ensure the antibody performs within acceptable parameters. This protocol describes the use of dapi to label nuclear dna of cells grown in culture. Our flow cytometry protocol has proven useful in all these scenarios 18, and similar approaches to detect rpa binding to chromatin have also been reported 19,20. The 5 mgml dapi stock solution may be stored at 26c for up to 6 months or at.
Kapuscinski described the use of dapi for dna analysis, identification of mycoplasma, and for staining a variety of biological materials. The chapter presents the staining protocol that has been developed for use in cell suspensions after fixation with 70% ethanol. The fixed cells should be analyzed as soon as possible. Harvest cells wash 2x in pbs to get rid of serum proteins. Flow cytometry hotz ma, gong j, traganos f, and darzynkiewicz z. The facility provides a wide variety of cell analysis and sorting services. The level of permeabilization is important as epitopes access may require different levels of permeabilization e. Livedead stain this protocol is for unfixable stains such as dapi, pi, and the sytox dyes 5 ml flow tubes. Dec 14, 2000 dapi has been widely used for flow cytometric determination of dna content and cell cycle analysis 2, 3, 7, 8. Dapi counterstaining protocols thermo fisher scientific us. Hoechst 33258 ethidium bromide for tracking the number of cell cycles. Cell cycle stainingdapi flow cytometry core facility. Comparison of the assays of in situ dna degradation and chromatin changes. Pdf live sorting and survival of unstained and dapistained.
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