The NUCLEAR-ID ® Red DNA Stain is a cell permeable dye, designed for use in a range of fluorescence detection technologies, in the discrimination of nucleated cells. It is resistant to photobleaching and is suitable for live-cell staining of nuclei. Also this dye provides a convenient approach for studying the induction and inhibition of cell cycle progression by flow cytometry. The amount of dye used in the protocol is good in live cells for 4+ days. Preparation: One first needs to make stock solution (10mM) for each dye. I have already aliquot Green dye in 20 0.5mL eppendorf tubes because the Green dye we ordered came in as one big vial (1mg), while red and far red came in as 20 different vials (50ug each).
It releases an orange fluorescence (605 nm) when excited by UV light (~300 nm). Ethidium bromide can also detect RNA depending on how much RNA folding occurs. Ethidium bromide is not a good probe for detecting DNA in live cells, however. this is because it is impermeable to intact cell membranes. Over the years, health concerns have arisen over ... DAPI, (pronounced as 'DAPPY') or 4′,6-diamidino-2-phenylindole, is a fluorescent stain that binds strongly to adenine–thymine rich regions in DNA.It is used extensively in fluorescence microscopy.As DAPI can pass through an intact cell membrane, it can be used to stain both live and fixed cells, though it passes through the membrane less efficiently in live cells and therefore the ... DNA Labeling in Living Cells Robert M. Martin,1 Heinrich Leonhardt,1,2 and M. Cristina Cardoso1* 1Max Delbr€uck Center for Molecular Medicine, Berlin, Germany 2Ludwig Maximilians University Munich, Department of Biology II, Planegg-Martinsried, Germany Received 26 January 2005; Revision Received 9 May 2005; Accepted 28 June 2005 Background: Live cell ﬂuorescence microscopy experi-
Figure 1. Cell cycle analysis using Vybrant DyeCycle Violet Stain. Histogram of live Jurkat cells stained with Vybrant DyeCycle Violet Stain showing DNA content distribution. G 0 /G 1 and G 2 /M peaks are separated by the S phase distribution. Violet 405 nm excitation was used with a 440/40 nm bandpass filter. Testing also indicates there is little or no genotoxicity on Syrian Hamster Embryo (SHE) cells, human lymphocytes, mouse lymphoma cells, or noted in the AMES test. The stain can be used with a blue-light transilluminator which causes less damage to the DNA being visualized and offers better efficiency for later cloning. Nuclear DNA staining (fluorescent probe) for FACs, high content screening, live cell studies and chromosome tracking Live MCF-7 breast cancer cell spheroid stained with SiR-DNA Live cell time lapse confocal imaging of a HeLa cell stained with SiR-DNA, note the very low background.
BrightCell™ Photostable Media: Live cell imaging cell culture media and supplements developed to protect cells from light-induced cellular damage.Low autofluorescence and photobleaching dramatically improves the quality of data that can be obtained during fluorescent live cell imaging experiments. Therefore, although SYTORNASelect does stain fixed cell nucleoli 16, 30, it may not be good for live cell nucleolar RNA imaging applications. Clearly, the RNA-selective dyes reported in this study surpassed commercially available RNA probes in terms of their usefulness in live cell imaging experiments. Dye Cell Tolerability Cell Cycle Analysis by Flow Cytometry: Flowing your Way through Life’s Cycle Over the past few decades the mammalian cell cycle has been well documented. Although there are lots of checkpoints as cells move through the cycle, we can very simply divide the cell cycle into three stages according to the DNA content in the nucleus.
DRAQ7™ is a live-cell impermeant, far-red emitting DNA dye for viability, sample quality, apoptosis and fixed cell nuclear counterstaining. DRAQ7™ has many cellular analysis applications and is highly compatible with existing protocols across a wide range of instrumentation platforms. However this live-cell method has relied on the use of the cell permeable Hoechst 33342 DNA-intercalating dye, limiting users to flow cytometers equipped with a UV laser. We have modified this protocol to use a newer live-cell DNA dye, Vybrant DyeCycle Violet, compatible with the more common violet 405nm laser.
Altogether DRAQ5 fulfils several of the requirements for a live cell DNA dye, i.e., it (a) is an easy to use cell‐permeant DNA dye, (b) is extremely photostable; (c) allows simultaneous imaging of the available genetically encoded fluorescent proteins, and (d) reflects accurately the spatial concentration of DNA in living cells. Cytoskeletal live cell imaging is extremely powerful when investigating cellular processes such as cytokinesis, motility and organelle transport and organization. The current experimental procedures remain nevertheless cumbersome and long. This post demonstrates how cell permeable, transfection free, Tubulin and Actin red fluorescent dyes help Cell biologists in analysing cytoskeleton dynamics ...
Figure 2. Live cell imaging of cellular lysosomes. A) HeLa cells expressing Mitochondria-GFP were stained with BioTracker 560 Orange Lysosome Dye (SCT019) and Hoechst DNA stain (Blue). B) Live HeLa cells stained with Live Cell Microtubule Stain (Blue) and BioTracker 540 Red Lysosome Dye (SCT141). What is a good nuclear stain for live imaging? I'm now needing to image cell cultures. I am thinking about transiently transfecting a fluorescent DNA marker, but I think it will be too sparsely ... One advantage of Hoechst 33342 is that it is membrane permeant and, thus, can stain live cells. Hoechst 33342 binds to adenine-thymine-rich regions of DNA in the minor groove. On binding to DNA, the fluorescence greatly increases. This protocol describes the use of Hoechst 33342 to label nuclear DNA of cells grown in culture.
For yeast and fungal cells we have developed a series of staining kits that combine the dead cell-specific Live-or-Dye™ stains with all-cell stains designed with yeast properties in mind. The Yeast Live-or-Dye™ Live/Dead Staining Kit combines a cell-permeant green dye, Thiazole Orange, which concentrates in the nucleus of live cells, with Live-or-Dye™ 568/583 (Fig. 5). A concentration of 0.1–12 μg/ml is commonly used to stain DNA in bacteria or eukaryote cells. Cells are stained for 1-30 min at room temperature or 37 °C and then washed to remove unbound dye. A green fluorescence of unbound Hoechst dye may be observed on samples which are stained with too much dye or which are washed partially. Basics of DNA Cell Cycle Analysis www.phoenixflow.com Page 2 This chapter is organized into progressively more advanced sections. Feel free to skip ahead to the level appropriate for your background.
Background. Live-cell fluorescence microscopy (LCFM) is a powerful tool used to investigate cellular dynamics in real time. However, the capacity to simultaneously measure DNA content in cells being tracked over time remains challenged by dye-associated toxicities. toxic, non -permeable DNA -binding dye. Viable cells exclude the dye while dead cells take up the dye, which becomes fluorescent upon binding to DNA. Both assays are non -toxic to cells, so viable cells remain in the sample well following measurement of the live or dead signals. In addition to providing real time kinetic
NucSpot® Live Cell Nuclear Stains are cell-membrane permeable DNA dyes that specifically stains nuclei in live or fixed cells. They have excellent specificity for DNA without the need for a wash step, and they have low toxicity for live cell imaging. This cell-permeant red-fluorescent dye stains the nuclear DNA of all cells in culture, regardless of viability, and can be used as a substitute for DAPI in imaging assays where nuclear staining is desired. With excitation at 622 nm and peak emission at 645 nm, Live Red Dye does not interfere with the FITC channel in imaging assays.
DRAQ5™ is a novel far-red fluorescent DNA dye that can be used in live cells in combination with other common fluorophores, especially GFP & FITC-tags. DRAQ5™ nuclear stain has many cellular analysis applications and is highly compatible with existing protocols across a wide range of instrumentation platforms. This brief tutorial demonstrates the use of the PureBlu Hoechst 33342 Dye with the ZOE™ Fluorescent Cell Imager for routine nuclear staining in fluorescence microscopy and cell imaging applications.
Cell tracers are frequently used in dye dilution assays to monitor cell proliferation using flow cytometry. Other trackers and tracers in a wide variety of colors provide efficient and sensitive methods for monitoring specific cells within a population by flow cytometry or imaging, in culture or in whole animals. Nuclear Green LCS1 is a fluorogenic, DNA-selective and cell-permeant dye for analyzing DNA content in living cells. The Nuclear Green LCS1 has its green fluorescence significantly enhanced upon binding to double-stranded DNA. It can be used in fluorescence imaging, microplate and flow cytometry applications. This DNA-binding dye might be used for multicolor analysis of live and fixed cells.
SiR-DNA (SiR-Hoechst*) is a far-red, fluorogenic, cell permeable and highly specific live cell DNA probe for fluorescence imaging of the nucleus and DNA. Kit contains 50 nmol SiR-DNA and 1 umol verapamil. Price excludes VAT and shipping Flow cytometry allows researchers to measure several physical characteristics of cells including its shape, size, and internal complexity. It has long been used in cell counting and cell sorting but more recently, researchers have been using flow cytometry in a broad range of applications including: biomarker detection, cell signaling, pathway screening, protein engineering, and systems biology.
SiR-Hoechst (SiR-DNA) is a far-red fluorescent DNA probe being used widely for time-lapse imaging of living cells that is reported to be minimally toxic at concentrations as high as 10–25 µM. We recently had some good experience with a new dye called Sir-Hoechst. It is a DNA stain excited in far-red and it's compatible with live cells. We used it to monitor and track cells over several ... PureBlu Hoechst 33342 Nuclear Staining Dye is compatible with fixed and unfixed cells. It exhibits a higher permeability for live cell membranes than 4',6-Diamidino- 2-phenylindole dihydrochloride (DAPI) and is optimal for live cell DNA staining. For research purposes only. Catalog # Description
DRAQ5™ is a cell permeable far-red fluorescent DNA dye that can be used in fixed or non-fixed/ live cells in combination with common labels such as GFP or FITC. As with any cell-permeant DNA intercalating probe, DRAQ5 may inhibit cell division in long-term assays and should be tested for any effect. Live Cell Imaging of the Actin Cytoskeleton Clink for PDF Version Recent advances in organic chemical synthesis have facilitated the ultimate aim of producing small cell-permeable compounds which can efficiently label the actin cytoskeleton and track its dynamic properties 1-3 .
Similar to the DNA binding dyes, the dead cells can be excluded by gating on the less stained population (live cells). Fig. 25. Protein binding viability dyes. A. Live cell with dye bound to surface primary amines. B. Dead cell with dye bound to surface and intracellular primary amines. A more straightforward approach to concurrently measure DNA content and cell surface immunofluorescence is to combine the staining of DNA in live cells (e.g., with Hoechst 33342) with surface immunophenotyping (Loken, 1980). As mentioned, however, in some cell types it is difficult to obtain high resolution of DNA content analysis after ...
It is critical to understand the degree of cell death in any flow cytometry assay and exclude those cells from the analysis. BioLegend provides DNA dyes, Propidium Iodide and 7- AAD, that enter and stain dead cells, but are impermeable to live cells for rapid, cost- effective analysis of unfixed cells. In cases where cell fixation is required, we now introduce fixable Zombie Aqua. Protocol B: Staining Live Cells with Calcein Dyes Calcein AM, Calcein Violet AM, and Calcein Blue AM labeling dyes cross the cell membrane easily, selectively labeling live cells for analysis by flow cytometry or fluorescent microscopy; apoptotic and dead cells with compromised cell membranes do not retain calcein. Co-staining with Annexin V or ... Recent advances have produced several live cell dyes useful for DNA content analysis using violet (405 nm), blue (488 nm) and green (532 nm) excitation sources with emission in visible wavelengths. We . separated by the S-phase distribution. present a new dye with near-infrared emission for DNA content analysis
Cell-permeable DNA stains are popular markers in live-cell imaging. Currently used DNA stains for live-cell imaging are either toxic, require illumination with blue light or are not compatible ... Live cell imaging has revolutionized how biologists study cells, proteins and a multitude of processes and molecular interactions. Live cell imaging techniques allow scientists to observe cell structures and processes in real time, and over time. The observation of dynamic changes provides more insight into the operations of a cell than a ...
There are several methods for analyzing live, dead, and apoptotic cells by flow cytometry. As cells die, the membrane becomes permeable. This allows for antibodies to penetrate the cells, which can now mimic live cells. For this and other reasons, it’s important to remove dead cells from further analysis during your flow cytometry experiments. In a previous study, we demonstrated that thanks to its ability to interact with DNA and its spectral properties MA could be used as a supravital DNA probe for fluorescence microscopy and flow cytometry including analyses of the cell cycle. In this study, we evaluated the suitability of MA as a DNA dye for time‐lapse microscopy and flow ...
BACKGROUND: Live cell fluorescence microscopy experiments often require visualization of the nucleus and the chromatin to determine the nuclear morphology or the localization of nuclear compartments. METHODS: We compared five different DNA dyes, TOPRO-3, TOTO-3, propidium iodide, Hoechst 33258, and DRAQ5, to test their usefulness in live cell ... Instead of binding to DNA they bind proteins so if they are added to a sample before fixation, live cells will bind some dye – there will be proteins present on the surface of the cell, but dead cells will bind much more as the dye can enter the cell and have access to the abundance of intracellular proteins. Product information: DRAQ7 containing Live Cell Impermeant DNA Dye - Gentaur.com - Product infoRead More