Nucred live 647 readyprobes reagent

Manufactured by Thermo Fisher Scientific

Sourced in United States

The NucRed Live 647 ReadyProbes Reagent is a cell-permeant nuclear stain that can be used to label the nuclei of live cells. It utilizes a far-red fluorescent dye to stain DNA, enabling visualization of the cell nucleus.

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Lab products found in correlation

Px330 lmna grna1, by Addgene (2 mentions) Bz x800e fluorescent microscope, by Keyence (2 mentions) Jetoptimus, by Polyplus Transfection (2 mentions) Hoechst 33258 dye, by Thermo Fisher Scientific (2 mentions) Alexa fluor 488 dye, by Thermo Fisher Scientific (2 mentions) Alexa fluor 594 phalloidin, by Thermo Fisher Scientific (2 mentions) C1 confocal microscope, by Nikon (2 mentions) Sp8 confocal microscope, by Leica (1 mentions) Cytation 3 imaging reader, by Agilent Technologies (1 mentions) Spinning disk confocal microscope, by Leica (1 mentions) Dmi8 microscope, by Leica (1 mentions) Fc blocking antibody, by BD (1 mentions) Cryotome, by Leica (1 mentions) Filipin 3 complex, by Merck Group (1 mentions) Eight well glass bottom slides, by Ibidi (1 mentions) A1r laser scanning confocal microscope, by Nikon (1 mentions) Summit software, by Beckman Coulter (1 mentions) Moflo astrios eq system, by Beckman Coulter (1 mentions) Version 10, by FlowJo (1 mentions) Calcein am, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

NucRed® Live 647 (11 citations) NucRed Live 647 ReadyProbes (2 citations) NucRed Live (2 citations)

17 protocols using nucred live 647 readyprobes reagent

Intracellular Localization of HPFN-FITC

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Confocal laser scanning microscopy (CLSM) was used to define the intracellular localization of fluorescein isothiocyanate (FITC)-labeled HPFN (designated as HPFN-FITC) in HeLa cells. To form HPFN-FITC conjugates, FITC was loaded into the HPFN. HeLa cells seeded in 60 mm-polystyrene plates (5 × 10⁵ cells per dish) were treated with HPFN-FITC (24 µg mL⁻¹) for 1 h, 3 h, and 6 h, and then stained with NucRed Live 647 ReadyProbes Reagent (Invitrogen, USA) for 15 min at 37 °C in the dark. The cells were washed twice with PBS (pH 7.4) and fixed with 4% paraformaldehyde for 30 min. The cells were then visualized using a Leica SP8 confocal microscope with 100/63× oil-immersion objective lens.

Tang T., Sun X., Xu X., Bian Y., Ma X, & Chen N. (2019). Development of hollow ferrogadolinium nanonetworks for dual-modal MRI guided cancer chemotherapy. RSC Advances, 9(5), 2559-2566.

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SARS-CoV-2 Spike Protein Interaction

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GFP1-10 and GFP11-expressing HEK293T cells were seeded separately in a 24-well plate. One day post-seeding, cells were transiently transfected using the calcium-phosphate precipitation method^{52 (link)}. GFP1-10 cells were co-transfected with increasing amounts (0, 8, 16, 32, 64, 125, 250, 500 ng) of pCG_IFTM1, pCG_IFITM2, pCG_IFITM3, and 250 ng of pLV-EF1a-human ACE2-IRES-puro. GFP11 cells were transfected with 250 ng of pCG_SARS-CoV-2-Spike C-V5 codon-optimized. Sixteen hours post-transfection, GFP1-10, and GFP11 cells were co-cultured in a poly-l-lysine-coated 24-well plate. Twenty-four-hour post-coculturing, cells were fixed with 4% PFA and cell nuclei were stained using NucRed Live 647 ReadyProbes Reagent (Invitrogen) according to the manufacturer’s instructions. Fluorescence imaging of GFP and NucRed was performed using a Cytation3 imaging reader (BioTek Instruments). 12 images per well were recorded automatically using the NucRed signal for autofocusing. The GFP area was quantified using ImageJ.

Prelli Bozzo C., Nchioua R., Volcic M., Koepke L., Krüger J., Schütz D., Heller S., Stürzel C.M., Kmiec D., Conzelmann C., Müller J., Zech F., Braun E., Groß R., Wettstein L., Weil T., Weiß J., Diofano F., Rodríguez Alfonso A.A., Wiese S., Sauter D., Münch J., Goffinet C., Catanese A., Schön M., Boeckers T.M., Stenger S., Sato K., Just S., Kleger A., Sparrer K.M, & Kirchhoff F. (2021). IFITM proteins promote SARS-CoV-2 infection and are targets for virus inhibition in vitro. Nature Communications, 12, 4584.

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Live-Cell Mobility Imaging of MECs

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Primary MEC^WT (GFP⁺ cells isolated from ACTA2-Cre^ERT2:ROSA-TG mice) and MEC^Lef-1KI (GFP⁻ cells isolated from ACTA2-Cre^ERT2:Lef-1KI^+/+ mice) were plated separately at 1×10⁵ cells per well on a 6-well dish. Cells were grown as described above. Living cell nuclei were labeled with NucRed Live 647 ReadyProbes Reagent (Invitrogen) by incubating the cells with two drops of reagent per milliliter of media for 30 minutes, and prior to imaging the media was replaced with fresh modified SAGM. Starting eight hours after seeding, live-cell mobility was recorded using a Leica spinning disk confocal microscope fitted with a CO₂ incubation chamber and 37°C heated stage. Images were collected using differential interference contrast (DIC) and a 630nm wavelength far red laser every five minutes for three hours.

Lynch T.J., Anderson P.J., Rotti P.G., Tyler S.R., Crooke A.K., Choi S.H., Montoro D.T., Silverman C.L., Shahin W., Zhao R., Jensen-Cody C., Adamcakova-Dodd A., Evans T.I., Xie W., Zhang Y., Mou H., Herring B.P., Thorne P.S., Rajagopal J., Yeaman C., Parekh K.R, & Engelhardt J.F. (2018). Submucosal Gland Myoepithelial Cells Are Reserve Stem Cells That Can Regenerate Mouse Tracheal Epithelium. Cell stem cell, 22(5), 653-667.e5.

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Carnoy's Fixation and Immunofluorescence Staining of Mouse Colon

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For Carnoy’s fixation, 1 cm of colon was dissected from the mouse and unfolded into a square shape. The tissues were fixed using Carnoy’s fixative solution (10% glacial acetic acid, 30% chloroform, 60% ethanol, and 1 g ferric chloride) and embedded in frozen section medium (Leica Biosystems, Nussloch, Germany) at −80 °C for 1 h before storage at –20 °C. For further staining, we sectioned blocks into 6 μm using a cryotome (Leica Biosystems), fixed them in precooled (−20 °C) acetone for 2 min, and washed them in fresh PBS for 5 min. The tissues were incubated with Fc blocking antibody (BD Biosciences, San Jose, CA, USA) for 30 min at room temperature and then with anti-Muc2 antibody (Abcam, Cambridge, UK) for 2 h at room temperature as well. After rinsing in fresh PBS, the tissues were incubated with Alexa Fluor 488-conjugated anti-IgG antibody (Abcam), counterstained with NucRed Live 647 ReadyProbes Reagent (Invitrogen, Carlsbad, CA, USA), and observed under a Leica DMi8 microscope (Leica Microsystems, Wetzlar, Germany).

Lee S.H., Seo D., Lee K.H., Park S.J., Park S., Kim H., Kim T., Joo I.H., Park J.M., Kang Y.H., Lim G.H., Kim D.H, & Yang J.Y. (2023). Biometabolites of Citrus unshiu Peel Enhance Intestinal Permeability and Alter Gut Commensal Bacteria. Nutrients, 15(2), 319.

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Cholesterol Quantification in Cultured Cells

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Cells were seeded on ScreenStar 96-well microplate and treated with indicated compounds 24 h later and fixed the day after. Samples were quenched with 50 mM NH₄Cl at RT for 15 min. Cholesterol staining was performed with 0.1 μg/ml filipin III complex (Sigma-Aldrich, Munich, Germany) diluted in PBS and 10% fetal calf serum at RT for 2 h. Cells were incubated with 2 drops/mL NucRed™ Live 647 ReadyProbes™ Reagent (Invitrogen, Waltham, Massachusetts, USA) in PBS at RT for 15–30 min to stain the nucleus. Detection of cholesterol was performed after washing using the ImageXpress Confocal HT.ai. Nine sites were imaged per replicate. Filipin III was detected at the DAPI channel and NucRed at the Cy5 channel. Image analysis was performed via MetaXpress.

Skorda A., Lauridsen A.R., Wu C., Huang J., Mrackova M., Winther N.I., Jank V., Sztupinszki Z., Strauss R., Bilgin M., Maeda K., Liu B., Luo Y., Jäättelä M, & Kallunki T. (2023). Activation of invasion by oncogenic reprogramming of cholesterol metabolism via increased NPC1 expression and macropinocytosis. Oncogene, 42(33), 2495-2506.

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TrkA Receptor Vesicle Dynamics

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NSC-34 control and MYO9A-depleted cells were seeded onto eight well glass bottom µ-slides (Ibidi) and transfected with the coding sequence of the TrkA receptor as in the recycling assay. After 24 h, a ROCK inhibitor (InSolution™ Y-27632—Calbiochem), was applied at 3nM in serum-free growth medium over-night and NucRed Live 647 ReadyProbes reagent (Invitrogen) was applied for visualization of nuclei. Cells were imaged using a Nikon A1R laser scanning confocal microscope over a period of ten minutes per cell, with z-stacks obtained. Resulting image series were then analysed using Imaris, with automatic detection of TrkA positive vesicles based on thresholding that was manually edited. Vesicles were then tracked by the software over-time and in 3D space to provide an output for speed the vesicles travelled, 10 cells were analysed per condition over three experimental repeats.

O’Connor E., Phan V., Cordts I., Cairns G., Hettwer S., Cox D., Lochmüller H, & Roos A. (2018). MYO9A deficiency in motor neurons is associated with reduced neuromuscular agrin secretion. Human Molecular Genetics, 27(8), 1434-1446.

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Quantitative Single-Cell DNA Analysis

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Flow cytometry data were analyzed and cells were isolated using a MoFlo Astrios EQ system (Beckman Coulter, Inc., Brea, CA), and FCS data were obtained using Summit Software (Beckman Coulter, Inc.) and then analyzed using the commercial software FlowJo version 10.2 (FlowJo, LLC, Ashland, OR). To determine the DNA contents in live cells, the cells were incubated in medium containing NucRed Live 647 ReadyProbes Reagent (#R37106, ThermoFisher Scientific Inc.), according to the manufacturer’s instructions.

Otsuka K, & Tomita M. (2018). Concurrent live imaging of DNA double-strand break repair and cell-cycle progression by CRISPR/Cas9-mediated knock-in of a tricistronic vector. Scientific Reports, 8, 17309.

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Adipose Progenitor Cell Transplant Assay

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For cell transplant assays, APs were isolated and transplanted in WAT as previously described (Jeffery et al. 2016 (link); Rodeheffer et al. 2008 (link)). For mTmG samples, NucRed^™ Live 647 ReadyProbes^™ Reagent (Thermo Fischer, R37106) was used as live/dead stain at 1:600 dilution. VWAT and SWAT of AP-ERαKO animals were pooled and GFP expression confirmed via FACS. Recipient male C57Bl/6J mice were anesthetized with isoflurane and surgeries performed using sterile technique. 0.5–1 million ERαKO AP cells were re-suspended in 15uL of PBS and injected into the left SWAT of 4–5-week-old congenic wildtype mice. A control PBS injection was done in the right SWAT. Mice were allowed to recover for 2 weeks, then placed on a HFD and treated with BrdU for 1 week. Left and right SWAT tissues were collected and analyzed separately via flow cytometry for incorporation with BrdU. ERαKO APs were identified by GFP fluorescence. Results were counted only for transplants in which more than 100 individual GFP-positive donor AP cells were recovered in the recipient SWAT.

Saavedra-Peña R.D., Taylor N, & Rodeheffer M.S. (2022). Insights of the role of estrogen in obesity from two models of ERα deletion. Journal of molecular endocrinology, 68(4), 179-194.

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Quantifying Spheroid Viability with Fluorescent Dyes

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Spheroids cultured in 96-well ultra-low attachment plates were treated with vehicle control (0.1% DMSO) or RDV (10 µM) with and without DEX (1 µM) for 48 h. After drug treatment, spheroids were incubated with a fluorescence dye-medium mixture containing NucBlue Live ReadyProbes Reagent, NucRed Live 647 ReadyProbes Reagent, or Calcein-AM (Thermofisher) for 30 min at 37°C as indicated by the manufacturer’s instructions. Fluorescence images were captured and analyzed using the Celigo Imaging Cytometer-4 Channels system from Nexcelom Bioscience LLC (Lawrence, MA). The dead/live ratio was quantified as the integrated intensity of the dead stain compared with the live stain using brightspot detection for each spheroid.

Liu K., Stern S., Heil E.L., Li L., Khairi R., Heyward S, & Wang H. (2023). Dexamethasone mitigates remdesivir-induced liver toxicity in human primary hepatocytes and COVID-19 patients. Hepatology Communications, 7(3), e0034.

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CRISPR-Mediated Gene Targeting Efficiency

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Cells grown on coverslips in six-well plates (5x10⁵ cells/well) were transfected with sgRNA plasmid targeting Lamin A (pX330-LMNA-gRNA1, addgene 122507) and donor plasmid (pCR2.1 Clover-LMNA Donor, addgene 122507) with 2.4 µg and 0.6 µg of DNA, respectively, using JetOptimus (Polyplus). Single vectors were transfected as negative controls. Cells were imaged alive 96 hr post-transfection using a Keyence BZ-X800E Fluorescent Microscope or were fixed with 1% PFA/2% Sucrose for 10 min and permeabilized with PBS-0.5% Triton buffer for 5 min. Gene targeting efficiency was determined by counting with Cell Profiler (Positive nuclei pipeline, threshold 0.25 for clover channel intensity) the percentage of Clover-positive cells using DAPI (Thermo Fisher 40 X objective, fixed cells) or NucRed Live 647 ReadyProbes Reagent (Thermo Fisher 20 X objective, live cells). Four independent experiments were performed and at least 500 cells were counted in each experiment. Data was plotted and represented in Graph Pad version 9.

Jimenez-Sainz J., Mathew J., Moore G., Lahiri S., Garbarino J., Eder J.P., Rothenberg E, & Jensen R.B. (2022). BRCA2 BRC missense variants disrupt RAD51-dependent DNA repair. eLife, 11, e79183.

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