Vybrant dyecycle violet staining protocol this basic protocol is optimized using jurkat cells suspended in complete medium rpmi10% fetal bovine serum and stained with vybrant dyecycle violet stain at 37. Cytometer is ideal for many cell research analysis protocols, including the nolyse, no wash and no cell loss protocol. This protocol simultaneously provides information about relative cell size, cell number, dna content and cell type via lineage tracing or tissue specific expression of fluorescent proteins in vivo cite this article. M vybrant dyecycle violet stain working solution prepared in step 2. Live cell cycle analysis of drosophila vybrantdyecycle. Dyecycle violet used for side population detection is a substrate of pa. The localization microscopy based on vdcv reported here shares some similarities with the results reported for smlm imaging of hoechst 33258, hoechst 33342 and dapi.
Uvactivated conversion of hoechst 33258, dapi, and. Optimized stem cell detection using the dyecycletriggered side. Dyecycle violet, ebfp, efluor 450, hoechst dyes, pacific blue, zombie violet viogreen alexa fluor 430, amcyan, bd horizon v500, bv510, cascade yellow, krome orange, pacific orange, qdot 525 zombie aqua. Quantitative superresolution localization microscopy of. The medical use of the dye has been largely superseded by more. The tagit violet proliferation and cell tracking dye should be stored at 20c upon receipt. Pdf studies are needed to understand the role of cd34 expressing cells with regard to. Violet 405 nm fluorescence channel blue blueviolet 45040 430470 pacific blue alexa fluor 405 popro1 dyecycle violet fixable violet dead cell stain celltrace violet calcein violet sytox blue fxcycle violet clickit pacific blue green blueviolet 52231 507537 qdot 525 horizon v500 fixable aqua dead cell. The bd horizon violet proliferation dye 450 vpd450 is functionally similar to cfse since they contain both an esterasecleavable group and an aminereactive succinimidyl ester group. The staining protocol is simple and includes incubating suspended cells in the presence of vybrant dyecycle stain and directly measuring the fluorescence without the need for any additional treatment or centrifugation steps. Hoechst 33258, dapi, and vybrant dyecycle violet are common fluorescent dyes used for staining dna and visualization of chromatin in cell nuclei by fluorescence and confocal microscopy. Crystal violet or gentian violet also known as methyl violet 10b or hexamethyl pararosaniline chloride is a triarylmethane dye used as a histological stain and in grams method of classifying bacteria. Vybrant dyecycle violet vdcv is a fluorescent dye with a predominant affinity for dna, and a weaker affinity for rna supplementary fig.
Hoechst 33258 or vybrant dyecycle violet are excited by uv and emit in the blue region of the light spectrum, thus leaving the reminder of the spectrum for detection of other cellular targets by probes emitting in green, yellow or red. Measuring apoptosis using annexin v and flow cytometry. We find that increasing the hoechst concentration from the standard live drosophila. However, because dcv has its excitation maximum shifted to a lower energy, it can still be sufficiently excited at longer wavelengths already falling into the violet range. Identification of side population cells from bladder cancer cells by. More recently, dyecycle violet dcv, a dna binding dye structurally similar to hoechst 33342 but with an excitation spectrum shifted toward the violet range, has also been used for sp analysis on flow cytometers equipped with violet laser diodes. Updating and significantly expanding upon the previous editions, basic cell culture protocols. Vybrant dyecycle violet dcv is another dnabinding fluorophore suitable for. Optimized stem cell detection using the dyecycletriggered. Abcg2mediated dyecycle violet efflux defined side population in. Vybrant dyecycle violet stain thermo fisher scientific.
Sytox aadvanced stain only stains necrotic cells with permeable membranes. Vybrant dyecycle violet stain v35003 from thermo fisher scientific. Fluorophore selection guide for the attune acoustic focusing. Live cell cycle analysis of drosophila vybrantdyecycle violet. At some point in their careers, virtually every scientist and technician, as well as many medical professionals, regardless of their area of specialization have a need to utilize cell culture systems.
The staining protocol is simple and includes incubating suspended cells in the presence of. Cell division is a process by which one cell produces two or more daughter cells. Authors accepted manuscript localization microscopy of dna in situ using vybrant dyecycle violet fluorescent probe. Cells were run on the attune cytometer using standard sensitivity at a flow rate of 100ulmin.
We have modified this protocol to use a newer livecell dna dye, vybrant dyecycle violet, compatible with the more common violet 405nm laser. Flow cytometry of the side population sp petriz 20. A protocol for cell cycle analysis of live drosophila tissues using the attune acoustic focusing cytometer is described. Pdf vybrant dyecycle violet stain discriminates two. In search for optimal imaging conditions for smlm, potentially in combination with structured illumination microscopy sim, a number of imaging buffers with various components were tested, including. Once the dmso is added to the tagit violet, use immediately, or store at 20c in a dry place and protected ifrom light, preferably in a dessicator or in a container with dessicant for no more than one month. Upon solubilization, the amount of dye taken up by the monolayer can be quantitated in a spectrophotometer or plate reader. Violet 405 nm excitation was used with a 44040 nm bandpass filter. The detailed experimental protocol for such combined measurements can be. Remove the vybrant dyecycle violet stain from the refrigerator and allow the vial to equilibrate to room temperature. Dyecycle violet used for side population detection is a.
I isolated primary adult mouse muscle stem cells these cells have an endogenous gfp reporter. Cd3cd28 stimulated cells were harvested on days as indicated. Localization microscopy of dna in situ using vybrant dyecycle. In the flow core, we have successfully obtained reproducible cell cycle data with the dna probes listed in table 1. A new approach to study nuclear nanostructure at single molecule resolution dominika zurekbiesiada, aleksander t. Crystal violet cell colony staining 1l fixingstaining solution. Bv 421 zombie violet azurite dapi efluor 450 calcein violet celltrace violet rulean fxcycle violet pacific blue sytox blue clickit plus edu pacific blue ebfp vibrant dyecycle violet horizon v450 fvd efluor 450, ecfp vioblue livedead fixable violet m turquoise alexafluor 405 dapi sirius pacific green livedead fixable aqua vgfp. Vibrant dyecycle violet in gibco hcec, indicating the presence of slow cycling cells in these cultures that reduced in population percentage 0. Uvactivated conversion of hoechst 33258, dapi, and vybrant. This protocol simultaneously provides information about relative cell size, cell number, dna content and cell type via lineage tracing or tissue specific expression of fluorescent proteins in vivo. Regain access you can regain access to a recent pay per article purchase if your access period has not yet expired. Crystal violet has antibacterial, antifungal, and anthelmintic properties and was formerly important as a topical antiseptic. Stem cell identification by dyecycle violet side population.
Fxcycle violet a vybrant dyecycle violet vybrant dyecycle green a a vybrant dyecycle orange a a cell permeable dye that can stain dna in both live or fixed cells. Violet chromatin condensationdead cell apoptosis kit with. Live cell cycle analysis of drosophila tissues using the attune acoustic focusing cytometer and vybrant dyecycle violet dna stain kerry flegel 1, dan sun 1, olga grushko 1, yiqin ma 1, laura buttitta 1. The protocol presented here allows for efficient cell cycle analysis coupled with cell type, relative cell size and cell number information, in a variety of drosophila tissues. This protocol describes the procedure for dcv staining as previously described mathew et al. In order to get a better understanding of stem cell biology and function, it is essential to reproducibly identify these stem cells from biological samples for subsequent characterization or isolation. This stain is often used when you need to count the number of cells e. Vybrant dyecycle violet is excited by the 405 nm violet laser, and is cell permeant. Technique for loading cells with bd horizon violet.
Vybrant dyecycle violet uorescence number of cells counted 0 50 100 150 200 250 500 0 1,000 1,500 2,000 dyecycle violet red uorescence dyecycle 0 200 400 600 800 1,000 escence 200 400 600 800 1,000 0 ordering information product cell status exem assays cat. Novel molecular probes reagents to analyze cellular function. Endometriosis is a disease that affects women and it is basically the mysterious process of the tissue from your uterus endometrial traveling around and ending up in pretty much any body part. We demonstrate, however, that the uvexcited dyes are not entirely stable when excited with the uv emitted by mercury arc lamps or 405 nm. Vybrant dyecycle violet stain is a cell permeable dna dye that can be used for. Im having issues with my dyecycle violet staining dcv. When this dye is loaded into hematopoietic cells, a sharply resolved side population was also observed, similar in appearance to that seen with hoechst 33342. Vybrant dyecycle violet stain is a cell permeable dna dye that can be used for cell cycle analysis and stem cell side population by flow cytometry. Oct 11, 2012 more recently, dyecycle violet dcv, a dna binding dye structurally similar to hoechst 33342 but with an excitation spectrum shifted toward the violet range, has also been used for sp analysis on flow cytometers equipped with violet laser diodes.
Dna staining for cell cycle university of kansas medical. Fluorophore selection guide for the attune acoustic. This is a simple assay useful for obtaining quantitative information about the relative density of cells adhering to multiwell cluster dishes. The stain will identify the somas of neurons a violet purple color, but not glial cells see figures 1 to 4. In our recent publications, we described the properties of these dyes using mass spectrometry and found a significant dependence of the abundances of the. Vybrant dyecycle violet fluorescence properties are altered by low ph or a high concentration of hydrogen peroxide. Proliferation as assessed by violet proliferation dye 450 vpd 450 experimental design. Quantitative superresolution localization microscopy of dna. The first is based on the simultaneous analysis of proliferation specific marker ki67 and cellular dna content, which discriminates restingquiescent cell populations g0 cell and quantifies cell cycle distribution g1, s or g2m, respectively. Mohana, hyunkeun lee, jeanyves roignant, udo birk, jurek w.
Vienna, austria in accordance with standard surface staining protocols. In general, any established labeling protocol used for flow cytometry will work with the imagestream see current protocols in cytometryfor general labeling techniques. Vpd450 is particularly suitable for multicolor applications where either green fluorescent proteins or fitclabeled antibodies are used. Dyecycle violet reagent has emission characteristics similar to those of hoechst 33342, but with a longer wavelength excitation maxima 369 nm. While bound to dna in nuclei of fixed cells, vdcv has an excitation maximum within the near uvrange and emits fluorescence in the blue region of the visible spectrum abs. Pbs, glycerol, mercaptoethylamine mea, glucose oxidase in combination with catalase and glucose, ascorbic acid and. Dyecycle violet convenient nuclear dyes as the remaining range of the visible spectrum can be used for detecting other subcellular targets, using dyes emitting in green, yellow, up to infrared.
Dcv is another dnabinding dye used as a violetexcited dye for vital cell cycle analysis. Optimization of imaging buffers for smlm of vybrant dyecycle violet. Cells were incubated at room temperature for 10 minutes. While bound to dna in nuclei of fixed cells, vdcv has an excitation maximum within the near uvrange and emits fluorescence in the blue region of the visible. Localization microscopy of dna in situ using vybrant. Vybrant dyecycle violet stains condensed chromatin more brightly than normal cell chromatin. Vybrant dyecycle violet stain thermo fisher scientific us. Therefore, dcv was evaluated in this study as a tool for identification of the sp from prostate cancer cell lines and from freshly harvested human prostate tissue. Isolation and applications of prostate side population cells based. Dyecycle violet reagent dcv is a dnabinding dye that can be used in the common violet laser diode vldequipped flow cytometer.
Abcg2mediated dyecycle violet efflux defined side population. In this unit, we describe two protocols for analyzing cell cycle status using flow cytometry. Kuperwasser lab crystal violet assay for proliferation aps. Stain cells on ice in the presence of azide when possible to reduce nonspecific capping of antibody. Dna staining for cell cycle university of kansas medical center. Vybrant dyecycle ruby stain thermo fisher scientific. The side population sp has become an important hallmark for the definition of the stem. M sytox aadvanced dmso solution to each tube containing 1 ml of cell suspension. Violet chromatin condensationdead cell apoptosis kit with vybrant dyecycle violet and sytox aadvanced 4 2. Unicellular organisms, like yeast, reproduce by cell division, whereas multicellular organisms, like us, use the same process to develop, grow, and maintain our tissues. Dyecycle violet dcv is a cell membrane permeable, fluorescent vital dye that intercalates into dna and is a substrate for abcg2mediated efflux. This is a pdf file of an unedited manuscript that has been accepted for. Pdf vybrant dyecycle violet stain discriminates two different. The validated dcv labeling protocol and sp analysis by flow.
Live cell cycle analysis of drosophila tissues using the. Tissue and cancer stem cells are highly attractive target populations for regenerative medicine and novel potentially curative anticancer therapeutics. Dyecycle violet dcv is a cell membrane permeable, fluorescent vital dye that. Dyecycle violet used for side population detection is a substrate of p. Fix and permeabilize cells using the foxp3 staining protocol. The stain will identify the somas of neurons a violetpurple color, but not glial cells see figures 1 to 4. This live cell stain allows the simultaneous table 1. Preciseaccurate cell cycle analysis in living cells safelow cytotoxicity for cell sorting and additional live cell experiments minimal compensationeasie.
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