Chromatin condensationdead cell apoptosis kit with. A combination of hoechst 33342 and propidium iodide have been extensively used for simultaneous flow cytometric and fluorescence imaging analysis of the stages of apoptosis and cellcycle distribution. Its uses are similar to hoechst 33342 h99 for counterstaining. Protoplasts from cell suspension cultures of haplopappus gracilis and solanum tuberosum cv. 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. Hoechst dyes are commonly used to stain genomic dna in the following applications. Introduction protocol for staining live cells protocol for staining fixed cells or tissue sections staining bacteria or yeast dye technical information ordering information introduction hoechst and dapi are popular blue fluorescent, nuclearspecific dyes that can be used to stain live or fixed cells.
Chemosensors free fulltext the use of hoechst dyes for. This product may be used in fluorescence microscopy, microplate, cuvette, and flow cytometry applications. Its uses are similar to hoechst 33342 h99 for counterstaining, apoptosis and cell cycle studies, but hoechst 33258 is reportedly less cellpermeant. The analysis and interpretation of dna distributions measured by.
Hoechst is a dna stain typically used to identify cell cycle profiles in live cells. Hoechst 33258 nucleic acid stain is a popular nuclear counterstain that emits blue fluorescence when bound to dsdna. Fixable dyes dead cells allow fixable viability dyes to cross their membranes. B2883 preparation instructions clear, yellow to brown.
Hoechst 33258, a dna binding dye is used to identify the nuclei of individual cells then the dhe fluorescence is quantified using the cellomic hcs machine to evaluate the oxidative stress. The primary laser at 488nm to excite the propidium iodide and the secondary laser at ultraviolet 351. This product may be used in fluorescence microscopy, microplate. Hoechst 33342 and pyronin y double staining can be used to measure dna and rna content in live cells by flow cytometry. Induction of apoptosis in human breast cancer cells via. Article pdf available in cold spring harbor protocols 20063 august 2006 with 3,504.
A second laser can be used to excite additional fluorochromes eg. The products may be used in fluorescence microscopy, microplate, cuvette, and flow cytometry applications. The hoechst 33342 dye has been used widely for staining the nuclei of living cells. Adjust the cell suspension to a concentration of 1 x 10 7 cellsml with cold. Ester is a suitable functional group for staining viable cells because it can pass. Hoechst 33258 ultra pure enz52402 enzo life sciences. This product may be used in fluorescence microscopy, microplate, cuvette and flow cytometry applications. Hoechst 33258 staining for detecting mycoplasma contamination.
The products may be used in fluorescence microscopy, microplate, cuvette, and flow cytometry. Box 48, 6700 aa wageningen the netherlands received january 14th, 1983 revision received may 21st, 1983 accepted may 21st, 1983 summary protoplasts. Large watercooled lasers can be adjusted to produce uv light. Introduction protocol for staining live cells protocol for staining fixed cells or tissue sections staining bacteria or yeast dye technical information ordering information. Adjust the cell suspension to a concentration of 1 x 10 7 cellsml with cold 4 o c pbsbsa buffer. The dyes have minimal fluorescence in solution, but become brightly. Common techniques that use dna, such as sequencing, cdna synthesis and cloning, rna transcription, transfection, nucleic acid labeling e. The use of hoechst dyes is reported in scientific literature for various applications including fluorescence microscopy, chromosome sorting, and cell cycle analysis using flow cytometry. Molecular structure of pureblu hoechst 33342 nuclear staining dye. Binding of hoechst 33258 to chromatin in situ stokke.
Dissolving hoechst dye in pbs is not recommended, but phosphatecontaining buffers may be used with dilute solutions of the dye. Protocol 7aad staining uv excitable dna dyes such as dapi and hoechst 33342 are normally excited with a uv laser 350360nm for cell cycle analysis. Moreover, their high affinity and specificity towards dna make hoechst dyes excellent targeting moieties, which can be conjugated to various other molecules in order to tether them to dna. This dye is often used to distinguish condensed pycnotic nuclei in apoptotic cells and for cell cycle studies in combination with brdu. Preparation of chromosome suspensions for flow cytometry. For each concentration we measured the average fluorescence as determined by flow cytometry, the. Bintje were stained with the dna specific bisbenzimidazole dye hoechst 33342 for flow. Hoechst 33258 is a possible carcinogen and possible mutagen. Hoechst 33258 is a fluorescent probe useful for detecting dna by fluorescence microscopy and flow cytometry. For instance, the intensity and lifetime of fluorescence of nuclei, stained by hoechst.
Preparation of cells for flow cytometry for further information. It can also be used to detect the contents of a sample dna by plotting a standard emissiontocontent curve. Hoechst 33258 staining dye solution ab228550 is a fluorescent stain for labeling dna in fluorescence microscopy. Add hoechst 33342 solution to each sample and incubate at 37c for 30 60 minutes. An example is the use of hoechst dyes to analyse how many cells of a population are in which phase of the cell cycle. This method is based on the detection of differences in chromatin condensation with hoechst 33342 as a probe and the detection of dead cells with propidium iodide as a probe for membrane damage. The fluorescence intensitybased ana lysis of hoechst 33258 and 7aaadstained images in. Moreover, their high affinity and specificity towards dna make hoechst dyes excellent. Is it possible to use hoechst 33258 as live dead marker. Dna staining of fixed and nonfixed plant protoplasts for.
Flow cytometry is increasingly employed as the method of choice for determination of nuclear dna content and ploidy level in plants. The becton dickinson flow cytometers do not calculate absolute cell counts total number of cells per sample. Is it possible to use hoechst 33258 as live dead marker with bv421, 605 and buv737 labeled antibodies for flow cytometry. Prelimi nary experiments show that this procedure can also be used for bivariate analysis of hu man and chinese hamster chromosomes. Hoechst 33258 dye is a possible carcinogen and possible mutagen. A number of macrocyclic dyes acridine orange derivative of acridines, hoechst 33342, hoechst 33258 derivatives of piperazine, ethidium, propidium derivatives of phenanthridine, etc. It can be used to examine cellular dna in most fluorescent applications.
By 1970 flow cytometry fcm measurement of the dna content of. Quantitation of dna using hoechst 33258 an essential element of cellular and molecular biology is the ability to quantitate dna in large numbers of samples at a sensitivity that enables determination of small amounts of sample. This method is based on the detection of differences in chromatin condensation with hoechst 33342 as a. Cell morphology was observed using an inverted microscope and hoechst 33258 staining. A flow cytometric method using hoechst 33342 and propidium. By using a dnaspecific stain, one can determine a dna profile e. Whole blood samples may be used undiluted unless the cell count is high, e.
Cell cycle analysis is a very common flow cytometry application. Propidium iodide staining of cells to assess dna c. Hoechst 33258 may be used on live or fixed cells and is also applicable for cell. However, the active compounds in cacf have not been investigated previously.
Pdf the use of hoechst dyes for dna staining and beyond. Dna fluorochrome staining with hoechst 33258 bisbenzimide is commonly used for detection of mycoplasma contamination in cell cultures. A flow cytometric method to detect apoptotic cells is described. Pureblu hoechst 33342 nuclear staining dye is a highly pure formulation of. Hoechst 33342 staining of viable cells for cell cycle. We recently reported antioxidant property of chloroform fraction of centratherum anthelminticum l. Hoechst 33258 and hoechst 33342 are the most used of these stains. The importance of this improvement for studies on chromosome damage caused by irradiation or mutagens is discussed. Hoechst 33342 hsc staining and stem cell purification protocol. Invitrogen hoechst 33342 nucleic acid stain is a popular cellpermeant nuclear counterstain that emits blue fluorescence when bound to dsdna. Ci c 3 h 3 c n n n o nh nh n h 2 n h ci n ci h 2 n hci n h ci n h fig. Morphological changes of cell apoptosis such as condensation of chromatin and nuclear fragmentations were found clearly.
In order to make that calcuation using becton dickinson flow cytometers, the total volume of cell sample fluid passing through the instrument during data acquisition must be determined. Flow cytometry protocol direct immunofluorescence of cells. Autofluorescence from endogenous cellular molecules such as the reduced forms of nicotinamide adenine dinucleotide or flavin adenine dinucleotide can interfere with imaging by. A number of macrocyclic dyes acridine orange derivative of acridines, hoechst 33342, hoechst 33258 derivatives of piperazine, ethidium, propidium. Hoechst 33342 bisbenzimide h33342 trihydrochloride is supplied as a lyophilized powder in 25 mg units. Hoechst 33342 protocol for imaging thermo fisher scientific. Intracellular staining flow cytometry tested during development recommended assay procedure. Viability staining protocol for flow cytometry bestprotocols.
We investigated the accuracy and precision of flow cytometric fcm estimates of bacterial abundances using 4. Quiescent cells at g0 phase have the same amount of dna as cells at g1 phase but lower rna levels compared to proliferating cells. Pdf hoechst dyes are among the most popular fluorophores used to stain. Hoechst 33258 staining dye solution ab228550 abcam. 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. Wear gloves and a mask, and work under a fume hood. Flow cytometric detection of g0 in live cells by hoechst. Protocols for hoechst staining the hoechst stains are fluorescent dyes used for labelling dna and rna through fluorescence microscopy and flow cytometry techniques. Thermo scientific hoechst 33342 24ethoxyphenyl54methyl1piperazinyl2,5bi1hbenzimidazole trihydrochloride trihydrate is a cellpermeable dna stain that is excited by. Flow cytometry protocol direct immunofluorescence of. The population should be separated into three groups. It thereby supplants previous methods that involved. Fluorescence microscopy and immunohistochemistry, often with other fluorophores. Hoechst 33258 may be used on live or fixed cells and is also applicable for cell cycle analysis and monitoring dna condensation by flow cytometry.
Prepare 1050 m hoechst dye solution with pbs or an appropriate buffer. Pdf flow cytometric analysis of brdusubsituted cells. There are available commercial dyes for excitation with a violet 405nm laser diode, however if cells are fixed with 4% fresh paraformaldehyde solution polysciences for 1 hour then labeled. The protocol recommended here follows that of bennett et al. For instance, the intensity and lifetime of fluorescence of nuclei, stained by hoechst 33258, were measured by microscopy in 8.
Quantitation of dna is a prelude to many practices in molecular biology. Photobleaching of hoechst 33258 is pronounced under the conditions of intense illumination, high magnification and resolution required for detection of mycoplasmas. The products may be used in fluorescence microscopy, micro. Staining of suspension cells with hoechst 33258 to detect apoptosis. The following is a general protocol for preparing assay solutions. A rapid method for measuring apoptosis and dualcolor.
In some applications, hoechst 33258 is significantly less permeant than hoechst 33342. Flow cytometric analysis of hela cell dna content right panel. Is it possible to use hoechst 33258 as live dead marker with. Hoechst dyes are among the most popular fluorophores used to stain dna in living and fixed cells. Quantitation of dna using hoechst 33258 an essential element of cellular and molecular biology is the ability to quantitate dna in large numbers of samples at a sensitivity that enables. Flow cytometric analysis of marine bacteria with hoechst 33342. Dapi staining of fixed cells for highresolution flow cytometry of nuclear dna. Flow cytometry cell cycle analysis using propidium iodide dna staining. Cell apoptosis was assessed by flow cytometry as well as dna fragmentation analysis. It can and will excite with both a uv and a strong violet laser and will read in a detr with a 45050bp filter. Hoechst 33258 is slightly more water soluble than hoechst 33342, but both have been used extensively to stain live cells. Protocol for dna quantitation using hoechst 33258 dye. Immunofluorescent staining of live cells for nuclear visualization 1. These dyes can also be used to detect the contents of a sample dna by plotting a standard emissiontocontent curve.
Labeling nuclear dna with hoechst 33342 csh protocols. Dapi and hoechst dye molecules attach at the minor groove of the. Hoechst 33342 staining dye solution ab228551 is a fluorescent stain for labeling dna in fluorescence microscopy. Flow cytometric analysis of brdusubsituted cells stained with 33258 hoechst. Hoechst 33342 staining of viable cells for cell cycle analysissorting. Hoechst 33342 hsc staining and stem cell purification. It thereby supplants previous methods that involved either fluorescence and absorption microspectrophotometry or chromosome counting, primarily because flow cytometry provides exceptional rapidity, convenience.
The binding of hoechst 33258 to rat thymocytes, human lymphocytes, and nhik 3025 tissue culture cells was studied by measuring the fluorescence and light scattering of the cells as functions of dye concentration using flow cytometry. The recent developments in the fields of microscopy and flow cytometry have sparked interest in such. After the incubation, analyze the stained cells by flow cytometry immediately, using uv488 nm dual excitaiton and measuring the fluorescence emission at 460 nm emission of hoechst. Plant science letters, 32 1983 7988 79 elsevier scientific publishers ireland ltd. Approximate fl uorescence excitationemission maxima, when bound to dna. Binding of hoechst with nucleic acids using fluorescence.
Staining cells with hoechst or dapi nuclear stains. Nuclear yellow hoechst s769121 is more commonly used as a neuronal retrograde tracer. Cell preparation for flow cytometry protocols bestprotocols. Hoechst 33342 staining dye solution ab228551 abcam. Hoechst dye is a known mutagen and should be handled with care. After the incubation, analyze the stained cells by flow cytometry immediately, using uv488 nm dual excitaiton and measuring the fluorescence emission at 460 nm emission of hoechst 33342 dye and 575 nm emission of propidium iodide.