Nucblue live reagent

Manufactured by Thermo Fisher Scientific

Sourced in United States

NucBlue Live Reagent is a fluorescent stain used to label nuclei in live cells. It binds to DNA, emitting a blue fluorescent signal when excited by light. The reagent can be used for visualization and quantification of live cells in various applications.

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12 protocols using nucblue live reagent

Live Cell Imaging of CAR-T Interaction

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Live cells were imaged with a DeltaVision microscope (GE Healthcare) and data were analyzed using ImageJ software. Raji and K562 cells were stained with Hoechst33342 (NucBlue® Live reagent, ThermoFisher) for 20 min, washed, and then co-cultured with CAR-T-cells for 10 min at 37°C.

Sommermeyer D., Hill T., Shamah S.M., Salter A.I., Chen Y., Mohler K.M, & Riddell S.R. (2017). Fully human CD19-specific chimeric antigen receptors for T-cell therapy. Leukemia, 31(10), 2191-2199.

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Visualizing ER and Nuclei in Cells

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Forty-eight hours after transfection with the plasmids, cells were treated with ER-Tracker Red (Thermo Fisher Scientific, Eugene, USA) and NucBlue Live Reagent (Hoechst 33342; Thermo Fisher Scientific, Eugene, USA). Microscopic images were obtained using a confocal microscope (Leica TCS SP8; Leica Microsystems, Mannheim, Germany).

Li L.X., Dong H.L., Xiao B.G, & Wu Z.Y. (2017). A Novel Missense Mutation in Peripheral Myelin Protein-22 Causes Charcot-Marie-Tooth Disease. Chinese Medical Journal, 130(15), 1779-1784.

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Imaging Kinetics of Transcription Factor

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A549 cells were seeded in 12-well culture plates and transfected with negative control siNC or siC/EBPβ. After 24 h, cells were stained with NucBlue^® live reagent (Hoechst 33342 dye, Thermo Scientific, Rockford, IL, USA) for 20 min following matufcturer’s protocol, then replaced with fresh culture medium. Images were taken by Operetta High Content Screening (HCS) System (PerkinElmer, Waltham, MA, USA) every 10 min for 48 h under 200× magnification.

Lee J.H., Sung J.Y., Choi E.K., Yoon H.K., Kang B.R., Hong E.K., Park B.K., Kim Y.N., Rho S.B, & Yoon K. (2019). C/EBPβ Is a Transcriptional Regulator of Wee1 at the G2/M Phase of the Cell Cycle. Cells, 8(2), 145.

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Antibody-based Nucleophosmin Detection

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Anti-B23/nucleophosmin antibody was purchased from Cell signaling technology (Danvers, MA). RNAse A was obtained from Qiagen (Hilden, Germany). 1-Step Human Coupled IVT kit, oleic acid, poly-A RNA, Prolong Gold with DAPI and Nucblue live reagent were purchased from ThermoFisher Scientific (Waltham, MA). Cytotoxicity LDH Assay Kit-WST and octanol were obtained from Wako pure chemicals (Japan). Cell Titer-Glo and Cell-tox glo kits were purchased from Promega (Madison, WI). TAMRA-labeled random 15mer RNA was synthesized by Fasmac (Japan). Anti-puromycin antibody was purchased from Millipore.

Kanekura K., Harada Y., Fujimoto M., Yagi T., Hayamizu Y., Nagaoka K, & Kuroda M. (2018). Characterization of membrane penetration and cytotoxicity of C9orf72-encoding arginine-rich dipeptides. Scientific Reports, 8, 12740.

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PRRT2 Expression Dynamics in HeLa Cells

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Growing in 35 mm glass‐bottomed dishes (Shengyou Biotechnology), HeLa cells were transfected with wild‐type or mutant PRRT2 expression plasmids. Forty‐eight hours after transfection, NucBlue Live Reagent (Thermo Fisher Scientific) and Alexa Fluor 594 wheat germ agglutinin (WGA; Invitrogene) were added and incubated for 20 and 10 minutes, respectively. After washing, cells were directly visualized under a confocal microscope (Leica TCS SP8; Leica Microsystems). The green‐fluorescent IOD (integrated optic density) of the whole cell and the cytoplasm was measured by ImageJ.

Zhao S., Li L., Chen Y., Chen Y., Liu G., Dong H., Chen D., Li H, & Wu Z. (2019). Functional study and pathogenicity classification of PRRT2 missense variants in PRRT2‐related disorders. CNS Neuroscience & Therapeutics, 26(1), 39-46.

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Live Cell Imaging of CAR-T Interaction

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Sommermeyer D., Hill T., Shamah S.M., Salter A.I., Chen Y., Mohler K.M, & Riddell S.R. (2017). Fully human CD19-specific chimeric antigen receptors for T-cell therapy. Leukemia, 31(10), 2191-2199.

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Time-Dependent Cell Adhesion Imaging

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Cells in media containing NucBlue Live Reagent (ThermoScientific) were plated at 5,000 cell/well in a 96-well plate for 15 m, 30 m, 45 m and 1hr total (3 replicates each) before the plate was inverted and tapped to remove media from the wells. Adherent cells were imaged using the EVOS FL Auto (ThermoScientific). As a control for total cells plated, separate wells were imaged at 3hr without media removal. Cell counts were generated using inForm 2.2 software (Perkin Elmer).

Dambal S., Baumann B., McCray T., Williams L., Richards Z., Deaton R., Prins G.S, & Nonn L. (2017). The miR-183 family cluster alters zinc homeostasis in benign prostate cells, organoids and prostate cancer xenografts. Scientific Reports, 7, 7704.

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Visualizing SOD1 Mutant Localization

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HEK293T cells were seeded on poly-D-lysine (PDL)-treated coverslips (NEST, China) and transiently transfected with various expression plasmids (pFLAG-SOD1-WT, pFLAG-SOD1-G17H, pFLAG-SOD1-E134*). Twenty-4 hours later, the cells were rinsed with 1 × phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde for 8 min, and then permeabilized with 1 × PBS supplemented with 0.01% Triton X100. The cells were blocked with 5% donkey serum and 1% bovine serum albumin (BSA; sigma, St. Louis, MO) in PBS for 1 h at room temperature. The primary antibody was rabbit antibody anti-flag (1:800, Cell Signaling Technology, 14793S). The secondary antibody was Alexa Fluor 488 donkey anti-rabbit. The cells were incubated with NucBlue Live Reagent (Hoechst 33342; Thermo Fisher Scientific, Oregon, United States) and visualized using Olympus FluoView FV3000 confocal microscopy with a ×63 objective.

Chen L.X., Xu H.F., Wang P.S., Yang X.X., Wu Z.Y, & Li H.F. (2021). SOD1 Mutation Spectrum and Natural History of ALS Patients in a 15-Year Cohort in Southeastern China. Frontiers in Genetics, 12, 746060.

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Synthesis and Characterization of Multimodal Nanoparticles

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Rhodium(III)
chloride hydrate (RhCl₃·xH₂O, Rh 38.5%–45.5%), Ruthenium(III)
chloride hydrate (RuCl₃·xH₂O, Ru 38%–40%), Ethylene glycol (EG, >99%), Poly(vinyl-pyrrolidone)
(PVP, 55 kDa), Ammonium heptamolybdate (AHM, (NH₄)₆Mo₇O₂₄·4H₂O), (3-Aminopropyl)triethoxysilane
(APTES, H₂N(CH₂)₃–Si(OC₂H₅)₃), Cy5.5 Mono NHS Ester (Cy5.5-NHS),
Triethylamine (TEA, (C₂H₅)₃N, ≥99%),
Dimethyl sulfoxide (DMSO, (CH₃)₂SO), Hydrochloric
acid (HCl, 37%), Tetraethyl orthosilicate (TEOS, Si(OC₂H₅)₄, ≥ 99%), Ethanolamine (EA, NH₂CH₂CH₂OH, ≥99%), Dulbecco’s
modified Eagle medium (DMEM), Fetal Bovine Serum (FBS), and Murine
macrophages (RAW 264.7, 91062702-1VL) were all purchased from Sigma-Aldrich.
Ethanol (EtOH, CH₃CH₂OH, 99.7%) was bought from
Solveco. All fluorescent probes, NucGreen Dead 488 ReadyProbes Reagent
(SYTOX Green), 4′,6-diamidino-2-phenylindole (DAPI), NucBlue
Live reagent (Hoechst 33342 dye), LysoTracker Green DND-26, Alexa
Fluor 555 Phalloidin, and Alexa Fluor 488 Phalloidin were all purchased
from ThermoFisher.

Saladino G.M., Vogt C., Li Y., Shaker K., Brodin B., Svenda M., Hertz H.M, & Toprak M.S. (2021). Optical and X-ray Fluorescent Nanoparticles for Dual Mode Bioimaging. ACS Nano, 15(3), 5077-5085.

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Quantifying Cell Viability with Calcein-AM and Hoechst

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After 48 h of dbcAMP treatment, the media were removed and 0.5 ml of pre-warmed fresh media containing 1 µM of Calcein-AM (Sigma-Aldrich) were added to each well and the plates were incubated at 37℃ for 30 min. To stain for cell nucleus and dead cells 1 drop of Hoechst3342 and 1 drop of Propidium Iodide (PI) were added into each well (NucBlue Live reagent, Thermo Fisher scientific). Cell survival was calculated as a percentage of green cells to the total cell number (Hoechst3342 positive, blue). In experiments where PI was included the cell viability was validated by counting the number of PI stained cells and calculating the percentage of dead cells (PI positive cells, red). PI staining was later on excluded since the results from the Calcein-AM/Hoechst-3342 matched very well with the results obtained from PI/Hoechst33-42 and Calcein-AM/PI (i.e., cells co-stained with PI/Hoechst (dead cells) were not Calcein-AM positive and cells costained with Calcein-AM/Hoechst3342 (live cells) were not PI positive).

Hansson M.L., Chatterjee U., Francis J., Arndt T., Broman C., Johansson J., Sköld M.K, & Rising A. (2021). Artificial spider silk supports and guides neurite extension in vitro. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 35(11).

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