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NUCLEAR-ID® Red DNA stain

Cell permeable DNA stain that can be used for a wide range of applications
 
ENZ-52406 200 µl 325.00 USD
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  • Far-red fluorescent specific DNA dye
  • Stable and highly pure
  • Live, permeable and fixed cells can be analyzed
  • No photobleaching effect
  • No RNase treatment is required
  • GFP and FITC compatible
  • UV laser source is not required for excitation
  • Validated for a wide range of cell densitites
  • Quick and easy to use!
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. Potential applications of this reagent for live-cell studies are in the determination of cellular DNA content and cell cycle distribution, for the detection of variations in growth patterns, for monitoring apoptosis, and for evaluating tumor cell behavior and suppressor gene mechanisms.

Product Specification

Quantity:200µl
 
Absorbance maximum:566nm
 
Emission maximum:650nm
 
Purity:≥93% (HPLC)
 
Quality Control:
  1. Absorption peak of NUCLEAR-ID® Red dye: λmax = 566 ± 4 nm
  2. % purity of NUCLEAR-ID® Red dye by HPLC: ≥93%
 
Appearance:Frozen liquid.
 
Applications:Flow Cytometry, Fluorescent detection
 
Shipping:Shipped on Dry Ice
 
Short Term Storage:-20°C
 
Long Term Storage:-80°C
 
Handling:Protect from light. Avoid freeze/thaw cycles.
 
Technical Info/Product Notes:The NUCLEAR-ID® Red DNA Stain is a member of the CELLESTIAL® product line, reagents and assay kits comprising fluorescent molecular probes that have been extensively benchmarked for live cell analysis applications. CELLESTIAL® reagents and kits are optimal for use in demanding imaging applications, such as confocal microscopy, flow cytometry and HCS, where consistency and reproducibility are required.
 
Protocol:A. Cell Staining with NUCLEAR-ID® Red Dye for DNA Cell Cycle Analysis by Flow Cytometry
  1. Treat cells with or without experimental compounds. Resuspend cells in appropriate buffer at a concentration of ≤ 5 x 105/mL in a test tube.
  2. Prepare NUCLEAR-ID® Red DNA Stain solution with media or common cell culture buffer of choice. A 250-fold to 500-fold dilution of NUCLEAR-ID® Red DNA Stain is recommended for live cell cycle DNA analysis. A 500-2000-fold dilution of NUCLEAR-ID® Red DNA Stain is recommended for ethanol-fixed or permeabilized cells.
  3. Prior to staining, collect the cells by centrifuging at 400 x g for 5 minutes. Carefully remove the supernatant.
  4. Re-suspend the cells in 0.5 mL freshly diluted staining solution.
  5. Gently mix and then incubate for 15-30 minutes at either room temperature or 37°C.
  6. Cells can be analyzed with flow cytometry directly without further treatment or washing.
  7. Analyze the samples in the PerCPCy5.5 or FL3 channel of a flow cytometer with a 488nm excitation laser. For 633 nm excitation, use appropriate collection filters >700 nm, FL4 or FL5, APC-Cy7 channel.
B. Live Cell Staining with NUCLEAR-ID® Red Dye for Nuclear Visualization by Fluorescence/Confocal Microscopy
  1. Adherent Cells. Grow adherent cells on cover slips inside a Petri dish filled with the appropriate culture medium. When the cells have reached the desired level of confluence, carefully remove the medium and dispense sufficient volume (~100 µL) of a 2000-fold to 4000-fold dilution (in media or buffer of choice) of NUCLEAR-ID® Red DNA Stain  to cover the monolayer of cells.

    Cell Suspensions. Centrifuge the cells for 5 minutes at 400 x g at room temperature (RT) to obtain a cell pellet. Then, carefully remove the supernatant by aspiration and dispense sufficient volume of a 2000-4000-fold dilution (in media or buffer of choice) of NUCLEAR-ID® Red DNA Stain to cover the dispersed cell pellet.

  2. Protect samples from light and incubate for 15-30 minutes at 37°C.
  3. Wash the cells with 100 µL buffer of choice, such as PBS. Remove excess buffer and place coverslip on slide. Analyze the stained cells by wide-field fluorescence or confocal microscopy (60X magnification recommended). Use a standard Rhodamine or Texas Red filter set for imaging the nucleus.
 
ENZ-52406-concentration
NUCLEAR-ID Red DNA Stain requires lower concentration than competitor’s dye to visualize dsDNA. HeLa cells were grown to ~60% confluency. Cells were stained with NUCLEAR-ID Red DNA Stain at a final concentration of 4000x or 2000x or with a competitor’s dye at an equivalent µM concentration at 37°C and gently washed post-staining. Cells were imaged at 15 min and 24h. Results show that 4000x NUCLEAR-ID Red DNA Stain was required for visualization of the dsDNA, while equivalent to 2000x was required for the competitor’s dye. At 24h, the competitor’s dye intensity and cell growth were dramatically reduced at the 2000x equivalent final concentration. At the same time point, 2000x of NUCLEAR-ID Red DNA Stain did not affect cell growth or fluorescent intensity. The NUCLEAR-ID Red DNA Stain shows lower cytotoxicity and requires lower concentration in live cell studies, resulting in lower costs.
ENZ-52406-cost-comare
Relative costs of using NUCLEAR-ID Red DNA in comparison to competitor dye in various applications: (A) Imaging (visualization), (B) Nucleated Cell Gating (flow cytometry) and (C) Live Cell Cycle analysis using flow cytometry. Dilutions can vary depending on cell strain and cell concentration. Notes: Assumes staining of a 100 µL staining volume Assumes staining of a 500 µL cell suspension volume Assumes a staining of 500 µL cell suspension volume
ENZ-52406 rev
NUCLEAR-ID Red DNA Stain requires lower concentration than competitor’s dye to visualize dsDNA. HeLa cells were grown to ~60% confluency. Cells were stained with NUCLEAR-ID Red DNA Stain at a final concentration of 4000x or 2000x or a competitor’s dye at the equivalent µM concentration at 37°C and gently washed post-staining. Cells were imaged at 15 min.

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ENZ-52406-concentration ENZ-52406-cost-comare ENZ-52406 rev

Product Literature References

Cellular IAP proteins and LUBAC differentially regulate necrosome-associated RIP1 ubiquitination: M.C. de Almagro, et al.; Cell Death Dis. 6, e1800 (2015), Abstract; Full Text
Plasminogen activator inhibitor-1 regulates tumor initiating cell properties in head and neck cancers: Y.C. Lee, et al.; Head Neck 38 Suppl 1, E895 (2015), Abstract;
Pervasive axonal transport deficits in multiple sclerosis models: C.D. Sorbara, et al.; Neuron 84, 1183 (2014), Abstract;
Zscan4 is regulated by PI3-kinase and DNA-damaging agents and directly interacts with the transcriptional repressors LSD1 and CtBP2 in mouse embryonic stem cells: M.P. Storm, et al.; PLoS One 9, e89821 (2014), Application(s): Flow cytometry measurements on mouse pluripotent cells, Abstract; Full Text
Epigenetic regulation of planarian stem cells by the SET1/MLL family of histone methyltransferases: A. Hubert, et al.; Epigenetics 8, 79 (2013), Application(s): Cell cycle analysis by flow cytometry, Abstract; Full Text
Wntless is required for peripheral lung differentiation and pulmonary vascular development: B. Cornett, et al.; Dev. Biol. 379, 38 (2013), Application(s): Detection of nuclei in lung cells by flow cytometry, Abstract;
Ionizing radiation induces mitochondrial reactive oxygen species production accompanied by upregulation of mitochondrial electron transport chain function and mitochondrial content under control of the cell cycle checkpoint: T. Yamamori, et al.; Free Radic. Biol. Med. 53, 260 (2012), Application(s): Cell cycle analysis by flow cytometry, Abstract;
Neurokinin 1 receptor mediates membrane blebbing and sheer stress-induced microparticle Formation in HEK293 Cells: P. Chen, et al.; PLoS One 7, e45322 (2012), Application(s): DNA nuclei staining by flow cytometry, Abstract; Full Text
Synthesis, cytotoxicity and cellular uptake studies of N3 functionalized Re(CO)3 thymidine complexes: M.D. Bartholomä, et al.; Dalton Trans. 40, 6216 (2011), Application(s): Nuclear DNA stain of human lung adenocarcinoma cells, Abstract;
A cell-permeant dye for cell cycle analysis by flow and laser-scanning microplate cytometry: Y.J. Xiang, et al.; Nat. Methods 6, an2 (2009), Application(s): Cell cycle analysis by flow cytometry, Abstract;

General Literature References

DNA measurement and cell cycle analysis by flow cytometry: R. Nunez; Curr. Issues Mol. Biol. 3, 67 (2001), Abstract;
Flow Cytometry. Methods in Cell Biology: Darzynkiewicz, Robinson and Crissman (eds.); 41 & 42, (1994), Book,

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