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. Cytometry protocol for using vybrant dyecycle violet. Novel high resolution imaging methods might be crucial for understanding of how the chromatin, a complex of dna and proteins, is arranged in the eukaryotic cell nucleus. This unit contains many helpful pointers to aid the user in obtaining the best possible results with these assays. Research article cytotoxicity and modes of action of the. Fluorescent dye optimization 4 optimization protocol for membrane impermeable dsdna dyes on incucyte flr andor zoom 1. The next generation in acoustic focusing cytometry. Using a 20,000 frame protocol and a 491 nm excitation, we detected very high numbers of fluorescent bursts that increased with the increasing concentration of the dye.
Optimized stem cell detection using the dyecycletriggered side. Quantitative superresolution localization microscopy of dna in situ. Localization microscopy of dna in situ using vybrant dyecycle violet fluorescent probe. Although this procedure seems obvious, we would like to stress two. This dye is a dnaselective, cell membranepermeant, and nonfluorescent stain that uses the violet laser for dna content analysis in living cells. Chemical modification of melphalan as a key to improving. A new approach to study nuclear nanostructure at single molecule resolution dominika zurekbiesiada, aleksander t. Subsequently, cells were measured in an lsrfortessa facs analyzer bectondickinson. Remove the vybrant dyecycle violet stain from the refrigerator and allow the vial to equilibrate to room temperature. Uvactivated conversion of hoechst 33258, dapi, and. Cytometer is ideal for many cell research analysis protocols, including the nolyse, no wash and no cell loss protocol. Mohana, hyunkeun lee, jeanyves roignant, udo birk, jurek w. Huss, phd department of pharmacology and therapeutics roswell park cancer institute buffalo ny outline. Live cell cycle analysis of drosophila tissues using the.
Flow cytometry of the side population sp petriz 20. Vybrant dyecycle violet stain thermo fisher scientific. Dyecycle violet dcv is a cell membrane permeable, fluorescent vital dye that intercalates into dna and is a substrate for abcg2mediated efflux. A new approach to study nuclear nanostructure at single molecule resolution. Dyecycle violet used for side population detection is a.
Vybrant dyecycle ruby cell staining protocol this staining protocol is optimized using jurkat cells suspended in complete medium rpmi10% fetal bovine serum and stained with vybrant dyecycle ruby stain at 37. Vybrant dyecycle violet stain discriminates two different. Basic protocol 1 flow cytometry to assess cell cycle status. A possible modification could be to substitute the hoechst 33342 with the new vybrant dyecycle violet stain invitrogen that has been optimized for the violet laser. 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. Measuring apoptosis using annexin v and flow cytometry. Seed a 96well microplate with cells at a density of 2,5005,000 cellswell so that cells are 2040% confluent at time of dye addition and incubate cells overnight at 37. 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. 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. Dear flowers, i have a user who wishes to measure cell cycle stage with a cocktail of labeled antibodies as hes interested in a. Pdf vybrant dyecycle violet stain discriminates two different.
Dyecycle violet used for side population detection is a substrate of p. Cytographswere analyzed using flowjo so ware celeza, switzerland. The stain can be excited by a 405 nm laser and used for cell cycle analysis and stem cell side population by flow cytometry. Vybrant dyecycle violet dcv is another dnabinding fluorophore suitable for sp. Flow cytometry purification of mouse meiotic cells protocol. Molecular probes vybrant dyecycle violet stain 200. However, the overall profile would not be expected to change drastically. Vybrant dyecycle violet stain vybrant dyecycle violet is used for dna content analysis in live cells. The red excitation and narrow emission of vybrant dyecycle ruby stain make it ideal for multiplexing due to the limited spectral overlap with other common dyes alexa fluor 488, fitc, and rpe and fluorescent proteins green fluorescent protein gfp. Molecular probes vybrant dyecycle ruby stain 400 assays. 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.
Vienna, austria in accordance with standard surface staining protocols. Vybrant dyecycle violet vybrant dyecycle green a a vybrant dyecycle orange a a cell. Hoechst 33342 h3569 dyecycle violet dye v35003 dyecycle green dye v35004 dyecycle orange dye v35005 dyecycle orange dye. We demonstrate, however, that the uvexcited dyes are not. Designed to maximize the potential of a flow cytometers violet laser, they not only show superior optical performance compared with many other 405 nm excited fluorochromes, but also significant. Preciseaccurate cell cycle analysis in living cells safelow cytotoxicity for cell sorting and additional live cell experiments minimal compensationeasie. The three lasers are spatially separated and are focused on the cuvette in three different locations. Vybrant dyecycle violet stain is dna selective, cell membranepermeant, and nonfluorescent until bound to doublestranded dna. The staining protocol is simple and includes incubating suspended cells in the presence of. Vybrant dyecycle violet stainwasmeasuredwith nmexcitation. Vybrant dyecycle violet stain from thermo fisher scientific. Quantitative superresolution localization microscopy of.
Optimized stem cell detection using the dyecycletriggered. Zurekbiesiada d, szczurek a, prakash k, lee hk, hagmann m, dobrucki jw, birk u, cremer c. Pdf vybrant dyecycle violet stain discriminates two. Vybrant dyecycle ruby stain thermo fisher scientific. Acoustic focusing cytometer to deliver a nowashnolyse protocol to minimize cell loss and simplify sample. Cytotoxicity and modes of action of the methanol extracts of. Cytometry protocol for using vybrant dyecycle violet with surface marker antibody staining. The purinergic receptor p2x5 regulates inflammasome. Cytotoxicity, mode of action and antibacterial activities.
Pdf studies are needed to understand the role of cd34 expressing cells with regard to. This is a pdf file of an unedited manuscript that has been accepted for. Functional cytomics josep carreras leukaemia research. More recently, a new assay based on the efflux of vybrant dyecycle violet stain dcv has been documented to discriminate sp cells. The stain can be excited by a 405 nm laser and used for cell cycle analysis and stem cell side population by. Localization microscopy of dna in situ using vybrant dyecycle. 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. Live cell cycle analysis of drosophila vybrantdyecycle. Vybrant dyecycle violet stain description precise accurate cell cycle analysis in living cells safe low cytotoxicity for cell sorting and additional live cell experiments minimal compensation easier multiplexing flexible simple, robust staining protocol view a selection guide for all products related to cell cycle analysis of fixed and live. Vybrant dyecycle violet stain has been shown to not only work for both live cells and fixed cells in cell cycle assays, but to identify stem cell side populations and early progenitors in mammalian hematopoietic tissues see figure. This is a major disadvantage of this dye, as uv light sources plus an adequate optical setup are expensive and not common components of flow cytometers.
We have modified this protocol to use a newer livecell dna dye, vybrant dyecycle violet, compatible with the more common violet 405nm laser. Fluorophore selection guide for the attune acoustic. Cells were incubated at room temperature for 10 minutes. Localization microscopy of dna in situ using vybrant. These spectral properties make hoechst 33258, dapi, and vybrant 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. Optimization of imaging buffers for smlm of vybrant dyecycle violet. The vybrant dio celllabeling solution is a dye delivery solution that can be added directly to normal culture media to uniformly label suspended or attached culture cells for use in cellcell fusion, cellular adhesion and migration applications.
Hoechst 33258 or vybrant dyecycle violet are excited by uv and emit in the blue region of the light spectrum, thus leaving the. Unlysed whole blood samples were stained with the dnaselective and cell membranepermeant vibrant dyecycle violet stain. Cells were run on the attune cytometer using standard sensitivity at a flow rate of 100ulmin. In this unit, we describe two protocols for analyzing cell cycle status using flow cytometry.
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