This dye is often used to distinguish condensed pycnotic nuclei in apoptotic cells and for cell cycle studies in combination with brdu. Hoechst 33342 is a premeable dna dye that binds preferentially to at base pairs. Hoechst 33342 nucleic acid stain is a popular cellpermeant nuclear counterstain that emits blue fluorescence when bound to dsdna. Add hoechst 33342 solution to each sample and incubate at 37c for 30 60 minutes. Intracellular staining flow cytometry tested during development recommended assay procedure. Hoechst 33342 protocol for hca instruments hoechst 33342. 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. Flow cytometry of murine spermatocytes current protocols. Fixed cell staining protocols for pi, 7aad, topro3 and dapi. Hoechstimdm media stored at 4oc imdm with 2% fbs, 1mm hepes and 1% penstrep. 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.
Hoechst 33258 staining dye solution ab228550 is a fluorescent stain for labeling dna in fluorescence microscopy. 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. Viable hoechst 33342syto 11picen protocol for apoptosis ref. They are used in fluores cence spectroscopy, microscopy and flow cytometry to. Hoechst 33342 side population identifi cation is a conserved. Change fsc and ssc to log scale and adjust threshold to 200 on bd lsrii excite at uv or 405nm wavelengths collect emission at 461 nm. Exclusion of hoechst 33342 dye is a characteristic common to stem cells, as well as chemotherapyresistant cancer cells. Store at 28c protected from light for up to 1 month. A paired sample ttest was used for the comparison between h342 and sybr14 stained population, and used to determine. Box 48, 6700 aa wageningen the netherlands received january 14th, 1983 revision received may 21st, 1983 accepted may 21st, 1983 summary protoplasts. Hoechst 33342 is also frequently used as a nuclear counterstain in fluorescence microscopy assays. Dna staining of fixed and nonfixed plant protoplasts for. W220 cell cycleanalysis a prerequisite for flow cytometry is, that cells should be in a single cell suspension.
Hoechst 33342 stainingforcellcycleanalysisoflivecells. Hoechst,33342,solution,ruo 561908 bd bioscienceseurope. Invitrogen hoechst 33342 nucleic acid stain is a popular cellpermeant nuclear counterstain that emits blue fluorescence when bound to dsdna. It can also be used to detect the contents of a sample dna by plotting a standard emissiontocontent curve. Protocols for purification of murine male germ cells by facs based on hoechst 33342 ho342 dye staining have been reported and optimized. The hoechst 33342 staining solution is a readytouse reagent for the identification of nucleated cells by flow cytometric analysis. Harvest bacteria add 10 m gml hoechst 33342 to 1ml of bacteria. This product may be used in fluorescence microscopy, microplate, cuvette, and flow cytometry applications. Hoechst 33342 is used for specifically staining the nuclei of living or fixed cells and tissues. Normally, these dyeexcluding cells can be sorted from enzymatically dissociated tissues with a uv cell sorter flow cytometer. A flow cytometric method using hoechst 33342 and propidium. Hoechst 33342 is widely used for enumeration of nucleated cells and for determination of dna content in cell cycle analysis. Hoechst 33342 staining for cell cycle analysis of live cells. Flow cytometric detection of g0 in live cells by hoechst.
Staining cells with hoechst or dapi nuclear stains. Dissolve 1 mg of hoechst 33342 powder molecular probes, eugene, or in 1 ml of distilled water or dmso. This stain is commonly used in combination with 5bromo2deoxyuridine brdu labeling to distinguish the compact chromatin of apoptotic nuclei, to. Flow cytometric analysis of hela cell dna content right panel. Likewise, when the staining process is over, the cells should be maintained at 4oc in order to prohibit further dye efflux. Highthroughput microtiter assay for hoechst 33342 dye uptake. Hoechst,33342,solution,ruo 561908 bd biosciencesus. Hoechst 33342 and pyronin y double staining can be used to measure dna and rna content in live cells by flow cytometry. This protocol describes staining and visualization of cells stained with hoechst 33342, but it. Both dyes have the absorption band at 350 nm and the emission band in the blue re gion.
Flow cytometric analysis of marine bacteria with hoechst 33342 article pdf available in applied and environmental microbiology 593. A flow cytometric method to detect apoptotic cells is described. Hoechst 33258 staining dye solution ab228550 abcam. Flow cytometric analysis of marine bacteria with hoechst 33342. Quiescent cells at g0 phase have the same amount of dna as cells at g1 phase but lower rna levels compared to proliferating cells. Binding of hoechst with nucleic acids using fluorescence. Chromatin condensationdead cell apoptosis kit with hoechst. Hoechst 33342 has also been used in a study to examine the effects of 9 calcium antagonists on abcg2bcrpmediated resistance and transport in hela and sn38resistant hela helasn100 cells, overexpressing abcg2bcrp. 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. Hoechst 33342 staining dye solution ab228551 abcam.
I need to check cell viability in a flow cytometry assay, and i always use pi. 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. Sep 01, 2016 analyzing cell death by nuclear staining with hoechst 33342. Analyse by flow cytometry collecting 25,000 events per. Flow cytometric characterization of viable meiotic and. Hoechst stains are part of a family of blue fluorescent dyes used to stain dna. Since the hoechst 33342 dye is specific for dna binding, ribonuclease treatment is not needed to avoid nonspecific rna staining. Hoechst 33342 mtg cmx rosamine protocol for apoptosis poot, m. Boisseau, claudine jalloustre, josy reiffers, philippe bernard, and francis lacombe.
Coefficients of correlation between the percentage of hoechst 33342 stained sperm h342. Intermittent blood flow in the kht sarcomaflow cytometry. Incubate at 37c for 10 minutes analyse 50,000 events by flow cytometry. Hoechst 33342 staining solution apoptosis and cell. Easytouse nuclear dye use pureblu hoechst 33342 dye for routine nuclear staining in cell imaging applications and fluorescence microscopy. Hoechst 33342 ready flow reagent is a bright, easytouse cellpermeant stain for cell cycle analysis on a flow cytometer.
Woolloongabba, brisbane, queensland 4102, australia. Plant science letters, 32 1983 7988 79 elsevier scientific publishers ireland ltd. Analyse by flow cytometry collecting 25,000 events per sample. This product may be used in fluorescence microscopy, microplate, cuvette and flow cytometry applications. In addition to its use in fluorescence microscopy and image analysis, hoechst 33342 is commonly used for flow cytometric applications, such as cell cycle analysis and stem cell side population identification. This staining is excellent for simultaneous detection of gfp expression. Single cell epithelial suspensions were extracted from these and incubated with hoechst 33342. The stain exits rapidly from the blood, with a halflife of 110 sec following an injection of. The fluorescent stain hoechst 33342, when injected i. 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. Hoechst 33342 staining dye solution ab228551 is a fluorescent stain for labeling dna in fluorescence microscopy. Propidium iodide pi, a redfluorescence dye excitationemission. Specimen source specimen status specimen type hoechst dye concentration.
Hoechst33342stainingforcellcycleanalysisoflivecells. Chromatin condensationdead cell apoptosis kit with. The administration of the fluorescent dna stain, hoechst 33342, to mice bearing the kht sarcoma, combined with flow cytometry, can be used to select cells according to their proximity to. Optimal staining time for murine samples is 90 min, whereas 120 min is optimal for human cells. Hoechst 33342 is a wellcharacterized blueemitting fluorescent compound widely utilized for nuclear staining. Immunofluorescent staining of live cells for nuclear visualization 1. Pdf livecell assay for detection of apoptosis by dual. Uv flow cytometry can be expensive, timeconsuming and not readily available to all laboratories. Kinetic analysis of intracellular hoechst 33342dna. A number of macrocyclic dyes acridine orange derivative of acridines, hoechst 33342, hoechst 33258 derivatives of piperazine, ethidium, propidium derivatives of phenanthridine, etc. However, the protocols are often challenging to follow, partly due to difficulties related to sample preparation, instrument. Spnonsp cell cycle status was established by hoechst 33342 and pyronin y staining. May be used for fluorescence microscopy or flow cytometry. Labeling nuclear dna with hoechst 33342 csh protocols.
Flow cytometric estimation of dna and rna content in. Flow cytometry and imaging, qimr berghofer medical research institute, herston, brisbane, queensland 4006, australia. Fluorescent dyes with aromatic amino or guanidine groups, such as propidium iodide pi, ethidium bromide eb, diaminophenylindole dapi. 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. Hoechst sp nonsp profi les were then generated by fl ow cytometry using standardized protocols. Im planning to stain fixed vero nuclei with hoechst 33342. Pi, analyzed by flow cytometry and fluorescence microscopy and expected values of each ratios, were made using linear regression analysis. Cell cycle tutorial queen mary university of london. This group renders hoechst 33342 a bit more hydropho bic than hoechst 33258. The optimal hoechst 33342 dye concentration and staining time may. Gibson, ll, singer, vl 1997 detection of apoptosis in live cells by mitotracker red cmxros and syto dye flow cytometry.
Hoechst 33342 is used for specifically staining the nuclei of living or fixed. Flow cytometric analysis of jurkat cells using phospho. Pureblu hoechst 33342 nuclear staining dye is a highly pure formulation of hoechst 33342 fluorescent dye figure 1 packaged in a userfriendly format. Pureblu hoechst 33342 nuclear staining dye is a highly pure formulation of. Analyzing cell death by nuclear staining with hoechst 33342.
Flow cytometer equipped with a uv laser and a 488 nm laser 4. Hoechst 33342 staining for side populations sue brusnahan. Quiescent cells at g0 phase have the same amount of dna as cells at g1 phase. Protocol 2 hoechst 33342 labeling of cells for flow cytometry of the side population this protocol describes the application of ho342 staining to identify and purify sidepopulation stem cells from different human and murine tissue sources. China and graduate school, the chinese academy of sciences, beijing. Livecell assay for detection of apoptosis by duallaser flow cytometry using hoechst 33342 and 7aminoactinomycin d article pdf available in nature protocol 21. Hoechst 33258 and hoechst 33342 are the most used of these stains. Hoechst 33258 is slightly more water soluble than hoechst 33342, but both have been used extensively to stain live cells. Pharmacokinetics, binding and distribution of hoechst 33342. The second is based on differential staining of dna and rna through costaining of hoechst 33342 and pyronin y, which is also useful to identify g0 cells from g1 cells. They are nonintercalating fluorescent probes for noncovalent labeling of dna. Hoechst 33342 is a cellpermeable fluorescent compound that is able to stain the dna of eukaryotic and. Nuclear yellow hoechst s769121 is more commonly used as a neuronal retrograde tracer.
I have used a combination of hoechst 33342 ho342 and pyronin y py to stain intact cells for dnarna content estimation with a dual source flow cytometer using uv and blue. Cell preparation for flow cytometry protocols bestprotocols. 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 mtg cmx rosamine protocol for apoptosis. Stable at room temperature convenient, readytouse formatno need to dilut. Hoechst 33342 can also be used to stain fixed cells by substituting hoechst 33342 for dapi in the protocol described in. Assaying cell cycle status using flow cytometry unit 28. Hoechst 33342 protocol for imaging thermo fisher scientific. Flow cytometry of the side population nyu langone health. Cell cycle analysis using hoechst 33342 in unfixed cells. Hoechst 33342 hsc staining and stem cell purification protocol. How do cell clumps affect quantitation of dna content. The protocol manual provides quite a range for both. Hoechst 33342pi double stain apoptosis detection kit genscript.
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. This dye emits blue fluorescence when bound to doublestranded dna with an emission maximum at 461 nm. It is available as a readytoreconstitute, highly pure powder form, with only one dilution step required to obtain a readytouse solution. Hoechst,33342,solution,ruo 561908 bd biosciencescn. Protocols for hoechst staining the hoechst stains are fluorescent dyes used for labelling dna and rna through fluorescence microscopy and flow cytometry techniques. Kinetic analysis of intracellular hoechst 33342 dna interactions by flow cytometry. We investigated the accuracy and precision of flow cytometric fcm estimates of bacterial abundances using 4.
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