Hoechst 33342 staining of viable cells for cell cycle analysissorting. The excitation and emission wavelengths of hoechst dna complex are 350 nm and 460 nm, respectively. Hoechst 33342 is a popular cellpermeant nuclear counterstain that emits blue fluorescence when bound to dsdna. Note that dapi has higher photostability than hoechst 33342. Hoechst 33342 efflux identifies a subpopulation of. Pureblu hoechst 33342 nuclear staining dye is a highly pure formulation of hoechst 33342 fluorescent dye figure 1 packaged in a userfriendly format. Staining dye solution ab228551 hoechst 33342 abcam. It can be used to examine cellular dna in most fluorescent applications. The ability of early hematopoietic progenitors to efflux certain fluorescent dyes such as rhodamine 123 and hoechst 33342 has long been appreciated and exploited for the isolation of these cells by fluorescenceactivated cell sorting facs.
Use pureblu hoechst 33342 dye for routine nuclear staining in fluorescence microscopy and cell imaging applications. Description the genscript double stain apoptosis detection kit hoechst 33342pi provides a rapid and convenient assay for apoptosis based upon fluorescent detection of compacted chromatin in apoptotic cells. Goodell et al18,21 have identified murine bone marrow cells with stem cell characteristics by their rapid efflux of the fluorescent vital dna binding dye, hoechst 33342. Use of hoechst 33342 staining to detect apoptotic changes. These bisbenzimides were originally developed by hoechst ag, which numbered all their compounds so that the dye hoechst 33342 is the 33,342nd compound made by the company. Measurement of cell proliferation in microculture using hoechst 33342 for the rapid semiautomated microfluorimetric determination of chromatin dna. Apoptosis, or programmed cell death, is a natural process of cellular selfdestruction. This stain can be used on both live and fixed cells 1.
Hoechst 33342 staining dye solution ab228551 is a fluorescent stain for labeling dna in fluorescence microscopy. The dnabinding bisbenzimide fluorochrome hoechst 33342 is being used routinely in radiobiological studies to assess cell kinetic parameters and tumour blood flow. Isolation of resident cardiac progenitor cells by hoechst 33342 staining article pdf available in methods in molecular biology clifton, n. Pureblu hoechst 33342 dye is a highly pure formulation of hoechst 33342 in a userfriendly format allowing easy preparation of the working solution, with no weighing step and only a single dilution after resuspension. Hoechst 33342 solution 20 mm thermo fisher scientific. Hoechst 33342 staining dye solution ab228551 abcam. Hoechst 33342 is a dye which binds to the at rich regions of the minor groove in. Harvest 1 ml of exponentially growing cells at max speed for 30 seconds. It is also important in embryogenesis, maintenance of immune tolerance, and development of the nervous system. Bisbenzimide tends to bind to adeninethyminerich regions of dna and can decrease its density. Add 2ugml hoechst 33342 solution to each sample at least 15 minutes before analysis. Hoechst 33342 is a specific fluorescent dna stain that binds within the minor groove of doublestranded atrich regions. Test principle hoechst 33342 is a premeable dna dye that binds preferentially to at basepairs.
Hoechst 33342 is a cellpermeable fluorescent compound that is able to stain the dna of eukaryotic and prokaryotic cells by binding with high affinity to the. Invitrogen hoechst 33342, trihydrochloride, trihydrate. In the present study, flow cytometric analysis and sorting demonstrate that these early damaged cells undergo the apoptotic process. The hoechst concentration, staining time, and staining temperature are all. Thermo scientific pierce hoechst 33342 fluorescent stain is a highquality solution of hoechst dye for fixed and livecell fluorescent staining of dna and nuclei in cellular imaging techniques. Thermo scientific pierce hoechst 33342 fluorescent stain is a highquality solution of hoechst dye for fixed and live cell fluorescent staining of dna and nuclei in cellular imaging techniques. In addition, the side population pattern is found to.
This means that cells may be flat, hard to see, and hard to analyze. Hoechst 33342 is a specific fluorescent dna stain that. Resuspend pellet in 1 ml buffered glucose plus 5 ugml hoechst 33342 dna dye 10 mgml stock solution. Hoechst 33342 is a cellpermeable fluorescent compound that is able to stain the dna of eukaryotic and prokaryotic cells by binding with high affinity to the minor groove of atrich dna sequences.
Analyzing cell death by nuclear staining with hoechst 33342. Identification of a cancer stem cell enriched side. The use of hoechst dyes for dna staining and beyond. Hoechst 33342 is used for specifically staining the nuclei of living or fixed cells and tissues. Stem cell identification and sorting using the hoechst 33342. This stain is commonly used in combination with 5bromo2deoxyuridine brdu labeling to distinguish the compact chromatin of apoptotic nuclei, to. Application a fluorescent dna stain qualified for use in mycoplama staining.
They bind to dna in the minor groove of polyat sequence rich. Hoechst dyes are cell membrane permeable and stain dna to emit intense blue fluorescence. Results incubation of hela cervical cancer cell with 5. The advantage of hoechst 33342 staining is the low cost of reagent and high degree of hsc enrichment, making the side population an appealing method to isolate hsc. It is available as a readytoreconstitute, highly pure powder form, with only one dilution step required to obtain a readytouse solution. This stain is commonly used in combination with 5bromo2. Staining with propidium iodide is a little more complicated because the dye also stains rna and an rnase. Richards wl, song mk, krutzsch h, evarts rp, marsden e, thorgeirsson ss. Hoechst 33342 staining of viable cells for cell cycle.
Because cells are not trypsinized or washed, they remain in their natural state. The excitation and emission wavelengths of hoechstdna complex are 350 nm and 460 nm, respectively. Fixation and permeabilization is not required for labeling cells, but physiologic conditions. Staining of live cells for dna content analysis by flow cytometry 1. Hoechst33342stainingforcellcycleanalysis oflivecells. Hoechst 33342 is a popular cell permeant nuclear counterstain that emits blue fluorescence when bound to dsdna. These dyes fluoresce at 461 nm when excited by ultraviolet light and can therefore be visualized using. Hoechst 33342 can also be used to stain fixed cells by substituting hoechst 33342 for dapi in the protocol described in labeling nuclear dna using dapi chazotte 2011a.
Apoptosis is necessary for routine cell turnover and tissue homeostasis. Unlike many other dna stains, it permits vital staining of at. Several related chemical compounds are used for similar purposes and are collectively called hoechst stains application. The blue fluorescent hoechst 33342 is a cell permeable nucleic acid dye usually used to identify chromatin condensation and fragmentation by staining the condensed nuclei of apoptotic cells. We present a simple and robust method to evaluate the transfection efficiency of commercially available transfection reagents intended to be established for use in nonmammalian cell lines. A method to evaluate the efficiency of transfection reagents in an adherent zebrafish cell line. Sep 01, 2016 during apoptosis the dna becomes condensed, but this process does not occur during necrosis. Nuclear condensation can therefore be used to distinguish apoptotic cells from healthy cells or necrotic cells.
The following procedure can be adapted for most cell types. Dna dye is known mutagen, handle with extreme care. Pdf isolation of resident cardiac progenitor cells by. It can also be used to detect the contents of a sample dna by plotting a standard emissiontocontent curve. Hoechst 33342, cas 23491523, is a high purity and quality chemical used as molecular probe and important as fluorescent stain for dna, chromosomes and nuclei fluorescent stain, dna, chromosomes and nuclei. Autofluorescence from endogenous cellular molecules such as the reduced forms of nicotinamide adenine dinucleotide or flavin adenine dinucleotide can interfere with imaging by. Hoechst 33342 casnumber 23491523 order from chemodex. In vitro morphological assessment of apoptosis induced by. Bisbenzimide hoechst 33342 is an organic compound used as a fluorescent stain for dna in molecular biology applications. Note that different concentration ranges for the hoechst dyes are suggested depending on the cell type see table 2.
Therefore, optimal resolution of the profile is obtained with great attention to the staining conditions. Hoechst 34580 possesses a dimethylamine group instead of the phenol, which shifts its emission maximum to 490 nm compared to the 461 nm of hoechst 33258 and hoechst 33342. Fluorochrome staining of nuclear dna allows one, through flowcytometric analysis, to estimate what pro portions of a cell population are in different phases of the cell cycle 67. Cell cycle analysis using hoechst 33342 in unfixed cells date. Hoechst 33342 is a cell permeable fluorescent compound that is able to stain the dna of eukaryotic and prokaryotic cells by binding with high affinity to the minor groove of atrich dna sequences. Hoechst 33342, a bisbenzimidazole, binds selectively to adenine. Use of hoechst 33342 staining to detect apoptotic changes in. Several related chemical compounds are used for similar purposes and are collectively called hoechst stains. It is found that the fluorescence emission of hoechst 33342 varies in low and high concentrations of dye, and the fluorescence of the cells shifts from blue to red with an increased dye intake, indicating a critical dye cell ratio for accurate hoechst 33342 staining petersen et al. This unit describes the use of hoechst 33342 to identify and purify murine hematopoietic stem cells, the so. Hoechst33342stainingforcellcycleanalysisoflivecells. Using hoechst 33342 staining, we observed increased numbers of apoptotic monocytes within 6 h of infection with. Each cell line examined was shown to require different hoechst 33342 concentrations and time of incubation with the dye to attain optimum fluorescence in the assay. Cells are incubated in ho at 37 degrees c for 3060 minutes.
In two human cell models cultured polymorphonuclear. The redfluorescent propidium iodide is a cell impairment dnabinding dye, which can only stain the cells in situations where there is increase in plasma. This dye is often used to distinguish condensed pycnotic nuclei in apoptotic cells and for cell cycle studies in combination with brdu. Chapter 9 supravital cell staining with hoechst 33342 and dioc53 harry a.
Growth medium, cell density, the presence of other cell types and other factors may influence staining. Basic protocol for staining cells the following procedure can be adapted for most cell types. Introduction the hoechst h0342 dyes are benzimidazole. Hoechst stains are part of a family of blue fluorescent dyes used to stain dna. In all cell lines, cell chromatinenhanced hoechst 33342 fluorescence was shown to be a linear function of the number of cells or cell nuclei per well when optimum assay conditions. Measurement of cell proliferation in microculture using. R233 dyes for living cell staining if the reagent is in a solid form, use dmso to prepare a solution with a certain concentration.
There are nuclei staining dyes that allow for viability of cells after staining. This stain is commonly used in combination with 5bromo2deoxyuridine brdu labeling to distinguish the compact chromatin of apoptotic nuclei, to identify replicating cells and to sort cells based on their dna content. Hoechst 33342 exhibits a 10 fold greater cell permeability than h 33258. The following is a simple procedure for determining cell number in preimplantation embryos. Functional isolation using hoechst 33342 the sp was first described in the bone marrow and muscle of various species, including rodents and humans 24, 25. Stem cell identification and sorting using the hoechst. It defines a population of cells that are able to actively pump the cytotoxic dye hoechst 33342 out of the cell. Hoechst dyeblue fluorescent stain specific for dna i. Invitrogen hoechst 33342 nucleic acid stain is a popular cellpermeant nuclear counterstain that emits blue fluorescence when bound to dsdna. Invitrogen hoechst 33342 nucleic acid stain is a popular cell permeant nuclear counterstain that emits blue fluorescence when bound to dsdna. Compared to hoechst 33258, hoechst 33342 is significantly more cellpermeable due to the addition of a lipophilic ethyl group and is usually preferred for living cell staining. Hoechst 33342 hsc staining and stem cell purification protocol. Cells are analysed without washing while in the media containing the ho.
Other dnabinding dyes can also be used including propidium iodide and ethidium bromide. Compared to hoechst 33258, hoechst 33342 is significantly more cell permeable due to the addition of a lipophilic ethyl group and is usually preferred for living cell staining. They bind to dna in the minor groove of polyat sequence rich areas. This kit contains two readytouse dyes bound to dna. Hoechst 33342 protocol for imaging thermo fisher scientific.
Assay procedure equilibrate all materials and prepared reagents to room temperature just prior to use and gently agitate. Chapter 9 supravital cell staining with hoechst 33342 and. Hoechst 33342 hsc staining and stem cell purification. H342 mitochondria staining dyescellstain mitored product code. The purpose of this study was to determine whether m.
Dyes that bind to dna, such as hoechst 33342 or 4,6diamidino2phenylindole dapi, can be used to observe nuclear condensation. Growth medium, cell density, the presence of other cell types and other factors may influence stain ing. Both hoechst 33342 and hoechst 33258 are watersoluble and stable in aqueous solutions. Hoechst 33342 bisbenzimide h33342 trihydrochloride is supplied as a lyophilized powder in 25 mg units. Hoechst33342stainingforcellcycleanalysis oflive cells. A flow cytometric method using hoechst 33342 and propidium. Using hoechst 33342 staining, we observed increased numbers of. Labeling nuclear dna with hoechst 33342 csh protocols. Double stain apoptosis detection kit hoechst 33342pi. Hoechst 33342, a type of bluefluorescence dye excitationemission maxima. The abcg2 transporter is an efficient hoechst 33342 efflux. Use pureblu hoechst 33342 dye for routine nuclear staining in cell imaging applications and fluorescence microscopy.
Nyulmc dart cytometry and cell sorting hoechst 33342 staining of viable cells for cell cycle analysissorting, nov2016 for cell cycle analysis and sorting of unfixed samples requires ultraviolet argon laser excitation, 450nm emission. Immunofluorescent staining of live cells for nuclear visualization 1. Likewise, when the staining process is over, the cells should be. Dna staining, hemopoietic stem cells, hoechst 33342, toxicity, cell cycle distribution fluorochrome staining of nuclear dna allows one, through flowcytometric analysis, to estimate what pro portions of a cell population are in different phases of the cell cycle 67. Hematopoietic stem cell characterization by hoechst 33342. As a dna minor groove binding compound, it can permeate cell membranes therefore preferentially stain nuclei figure 1. Invitrogen hoechst 33342, trihydrochloride, trihydrate, 100. This product may be used in fluorescence microscopy, microplate, cuvette, and flow cytometry applications. Request pdf analyzing cell death by nuclear staining with hoechst 33342 the nuclei of healthy cells are generally spherical, and the dna is evenly distributed. The optimal hoechst dye concentration and staining time for different. Protocol to count cell number of preimplantation embryos. For this reason we counter stain the cell with hoechst while simultaneously staining for annexinv and propidium iodide pi. Hoechst 33342 binds preferentially to adeninethymine at regions of dna.
Add hoechst 33342 solution to each sample and incubate at 37c for 30 60 minutes. Thermo scientific hoechst 33342 24ethoxyphenyl54methyl1piperazinyl2,5bi1hbenzimidazole trihydrochloride trihydrate is a cellpermeable dna stain that is excited by ultraviolet light and emits blue fluorescence at 460490 nm. Three properties of the dye contribute to the ability to distinguish stem cells in this way. The simultaneous display of the fluorescent staining of cells exposed to hoechst 33342 at 2 different wavelengths identifies a small and distinct subpopulation termed the side population sp. Radiation sensitivity of tumour cells stained in vitro or. The optimal cell density, concentration of hoechst 33342, and stain time for dna content analysis may vary by cell type. The staining pattern resulted from the simultaneous use of these dyes makes it possible to distinguish normal, apoptotic, and dead cell populations by flow. Hoechst 33342 is a wellcharacterized blueemitting fluorescent compound widely used for nuclear staining. Hoechst stains property may allow fluorescenceactivated cell and chromosome sorting according to the extent of dna synthesis which may provide a bridge between biochemical and cytologic analyses of processes related to dna replication.
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