Click it edu alexa fluor 647 kit

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Click-iT EdU Alexa Fluor 647 kit is a reagent used for the detection and quantification of DNA synthesis in proliferating cells. It incorporates the modified nucleoside EdU (5-ethynyl-2′-deoxyuridine) into DNA during active DNA synthesis. The incorporated EdU can then be detected using a copper-catalyzed click reaction with the Alexa Fluor 647 fluorescent dye.

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Alexa Fluor 647 (1520 citations) Alexa 647 (424 citations) Click-iT EdU (80 citations) Alexa Fluors (21 citations) Click-iT EdU Alexa Fluor 647 (12 citations) Click-iT Edu Alexa Fluor (5 citations) Click-iTTM EdU Alexa FluorTM 647 Flow Cytometry Assay Kit (3 citations) EdU Alexa Fluor Kit (2 citations) Click-iTTM Plus EdU Alexa FluorTM 647 flow cytometry assay kit (2 citations)

32 protocols using click it edu alexa fluor 647 kit

EdU Labeling and Immunostaining of Zebrafish

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Zebrafish were incubated in 0.5 mM EdU in egg water/0.5% DMSO (four animals/500 µl in 24-well plates) for 24 h (from 56 to 80 hpf and from 78 to 102 hpf). They were fixed in 4% paraformaldehyde and incubated in Click-iT EdU Alexa Fluor 647 Kit (Thermo Fisher Scientific) for 30 min. They were then processed for immunostaining with anti-GFP, anti-mCherry, and DAPI using the procedure described above.

Gunawan F., Gentile A., Fukuda R., Tsedeke A.T., Jiménez-Amilburu V., Ramadass R., Iida A., Sehara-Fujisawa A, & Stainier D.Y. (2019). Focal adhesions are essential to drive zebrafish heart valve morphogenesis. The Journal of Cell Biology, 218(3), 1039-1054.

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EdU Incorporation in Cerebral Organoids

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GBOs were incubated in GBO medium containing 1 μM EdU (Thermo Fisher Scientific) for one hour on an orbital shaker rotating at 120 rpm within a 37°C, 5% CO₂, 90% humidity sterile incubator. GBOs were fixed and processed for immunohistology at 1-hourand 2-week time points. EdU incorporation was detected using Click-iT EdU Alexa Fluor 647 kit (Thermo Fisher Scientific) (Berg et al., 2019 (link)).

Jacob F., Salinas R.D., Zhang D.Y., Nguyen P.T., Schnoll J.G., Wong S.Z., Thokala R., Sheikh S., Saxena D., Prokop S., Liu D.A., Qian X., Petrov D., Lucas T., Chen H.I., Dorsey J.F., Christian K.M., Binder Z.A., Nasrallah M., Brem S., O’Rourke D.M., Ming G.L, & Song H. (2019). A Patient-Derived Glioblastoma Organoid Model and Biobank Recapitulates Inter- and Intra-tumoral Heterogeneity. Cell, 180(1), 188-204.e22.

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Quantifying VIC Proliferation in Hydrogels

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The proliferation of VICs as a function of hydrogel substrate stiffness organization was monitored in both 1% and 15% FBS, corresponding to conditions for low and high levels of VIC proliferation. To quantify proliferation, 10 μM EdU in PBS (ThermoFisher, Cat# C10340) was added to the culture at 36 h and allowed to incubate for another 36 h before fixation. The samples were subsequently rinsed in 3% BSA twice, followed by permeabilization in 0.1% TritonX-100 in PBS for 1 h. After rinsing with 3% BSA, samples were incubated with the Click-iT reaction cocktail prepared from the Click-iT EdU Alexa Fluor 647 kit (ThermoFisher, Cat# C10340) for 30 min at room temperature. Samples were washed once with 3% BSA and once with PBS. After this, immunostaining was continued as described before. Mitomycin C (Sigma-Aldrich, Cat# M4287) was added at a concentration of 10 μg/mL 24 h after seeding during the proliferation inhibition experiments. After incubation with Mitomycin C for 2 h, two 1 h washes with 1% FBS medium without Mitomycin C were performed to wash away the excess Mitomycin C.

Ma H., Killaars A.R., DelRio F.W., Yang C, & Anseth K.S. (2017). Myofibroblastic activation of valvular interstitial cells is modulated by spatial variations in matrix elasticity and its organization. Biomaterials, 131, 131-144.

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Muscle Stem Cell Proliferation Assay

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For LRC/nonLRC experiments, muscle stem cells were isolated as described above from three H2B-GFP^+/−; rtTA^+/− mice (4 months old, labeled developmentally as above) and cultured in plating media (DMEM, 10% horse serum) for 50 h. EdU (Carbosynth) was pulsed into the media at 12 h and 2 h (10 µM final concentration) prior to fixation with 4% PFA for 15 min. Staining followed the Click-iT EdU Alexa Fluor 647 kit protocol (Thermo Fisher Scientific). For Pax3⁺/Pax3⁻ experiments, Pax3⁺ and Pax3⁻ MuSCs from forelimbs and pectoralis muscles were collected by FACs from Pax3-GFP/⁺ adult mice, plated on ECM-coated chamber-slides and cultured in high-serum conditions (20% FBS, F10) for 42 h. EdU (10 µM) was added during the final 12 h. EdU was detected by fluorescence microscopy. We tested the significance of EdU incorporation using logistic regression models and the Wald test (supplementary Materials and Methods).

Kimmel J.C., Hwang A.B., Scaramozza A., Marshall W.F, & Brack A.S. (2020). Aging induces aberrant state transition kinetics in murine muscle stem cells. Development (Cambridge, England), 147(9), dev183855.

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Labeling Proliferating Cells in Zebrafish Embryos

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Gata1 mutants and controls were transferred to 0.5 mmol/L EdU in egg water/0.5% dimethyl sulfoxide (DMSO) from 74 to 98 hpf. For FK506 experiments, EdU was added to media already containing DMSO or FK506 to reach a final concentration of 0.5 mmol/L EdU. The larvae were then fixed with 4% PFA for 3.5 hours at room temperature and washed with 1x PBST. The pericardial cavity was pierced using an ultrafine needle, the tails cut, and the entire larvae permeabilized overnight at 4°C in 1x PBST containing 1% Triton-X 100. Detection of EdU was performed using a Click-iT EdU Alexa Fluor 647 Kit (Thermo Fisher Scientific), where larvae were incubated in the Click-iT reaction cocktail for 1 hour. Embryos that show no staining in the heart and surrounding tissues were excluded from analysis.

Chow R.W., Fukui H., Chan W.X., Tan K.S., Roth S., Duchemin A.L., Messaddeq N., Nakajima H., Liu F., Faggianelli-Conrozier N., Klymchenko A.S., Choon Hwai Y., Mochizuki N, & Vermot J. (2022). Cardiac forces regulate zebrafish heart valve delamination by modulating Nfat signaling. PLoS Biology, 20(1), e3001505.

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Cell Proliferation Quantified by EdU

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Cells were pulsed with 10 μM EdU for 60 min, and fixed in 4% paraformaldehyde. EdU incorporation was assessed with the Click-iT EdU AlexaFluor 647 kit (ThermoScientific), following manufacturer's instructions. At least 20,000 cells were acquired using a BD Canto flow cytometer and analysed with FlowJo software.

Grey W., Ivey A., Milne T.A., Haferlach T., Grimwade D., Uhlmann F., Voisset E, & Yu V. (2018). The Cks1/Cks2 axis fine-tunes Mll1 expression and is crucial for MLL-rearranged leukaemia cell viability. Biochimica et Biophysica Acta, 1865(1), 105-116.

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EdU Labeling and Quantification in Tumor Sections

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Two hours prior to sacrifice, animals were treated with 25 mg/kg EdU via intraperitoneal injection. Tumor sections were prepared and treated according to the immunohistochemistry protocol through antigen retrieval. EdU was detected using the Click-It EdU Alexa Fluor 647 Kit (Thermo fisher) followed by incubation with 100ng/mL DAPI for 10 minutes at room temperature. The slides were mounted with ProLong Gold mounting medium (Thermofisher). Alexa 647 and DAPI were imaged using the Rockefeller University Bioimaging Resource Center and the images were processed using imageJ.

Maguire O.A., Ackerman S.E., Szwed S.K., Maganti A.V., Marchildon F., Huang X., Kramer D.J., Rosas-Villegas A., Gelfer R.G., Turner L.E., Ceballos V., Hejazi A., Samborska B., Rahbani J.F., Dykstra C.B., Annis M.G., Luo J.D., Carroll T.S., Jiang C.S., Dannenberg A.J., Siegel P.M., Tersey S.A., Mirmira R.G., Kazak L, & Cohen P. (2021). Creatine mediated crosstalk between adipocytes and cancer cells regulates obesity-driven breast cancer. Cell metabolism, 33(3), 499-512.e6.

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Cell Proliferation Analysis via EdU Assay

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EdU assay was performed using the Click-IT EdU Alexa Fluor 647 kit (Thermo Fisher, Waltham, MA, USA) as previously described (37 (link), 38 (link)). Apollo staining and DAPI staining for nuclei staining were performed. Under a fluorescence microscope, the blue fluorescence represents the nucleus stained by DAPI, and the red fluorescence represents the newly synthesized DNA stained by EdU. The incorporation rate of EdU was equal to the ratio of EdU-positive cells (red)/DAPI-positive cells (blue).

Sun J., Xu H., Lei Z., Li Z., Zhu H., Deng Z., Yu X., Jin X, & Yang Z. (2022). The lncRNA CASC2 Modulates Hepatocellular Carcinoma Cell Sensitivity and Resistance to TRAIL Through Apoptotic and Non-Apoptotic Signaling. Frontiers in Oncology, 11, 726622.

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Quantifying S-Phase Endothelial and Hematopoietic Cells

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Click-iT EdU Alexa Fluor 647 kit (Thermo Fisher, C10340) was used to analyse S phase endothelial and positively expressing kdrl+cmyb+ cells in the 32 hpf AGM. Embryos were injected at 32 hpf with 10 mM EdU staining solution^{76 (link)} into the sinus venosus and incubated for 5 min at 28 °C, followed by 4% paraformaldehyde overnight fixation at 4 °C. Embryos were washed three times for 5 min with PBSTw and placed in cold 100% acetone at −20 °C for 7 min and rinsed with dH₂O. Subsequently, embryos were permeabilized with 1% DMSO, 1% Triton in 1× PBS for an hour and washed three times for 5 min with PBSTw and then incubated with reaction cocktail (1× reaction buffer, CuSO₄ solution, Alexa Fluor azide and reaction buffer additive) for 1 h at room temperature in the dark. Samples were rinsed five times for 5 min with PBSTw, and then stained as stated above by IF with the addition of DAPI staining (1:500).

Ghersi J.J., Baldissera G., Hintzen J., Luff S.A., Cheng S., Xia I.F., Sturgeon C.M, & Nicoli S. (2023). Haematopoietic stem and progenitor cell heterogeneity is inherited from the embryonic endothelium. Nature Cell Biology, 25(8), 1135-1145.

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Zebrafish Immunofluorescence and EdU Assay

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Immunofluorescence of zebrafish larvae and confocal analysis were performed as previously described. The primary antibodies used for this study were anti-GFP (1:500; Aves Labs GFP-1020), anti-insulin (1:100; custom made by Cambridge Research Biochemicals), anti-Nkx6.1 (1:50; DSHB), and anti-Folr1 (1:50; Aviva Systems Biology-ARP41427_P050).
For measuring EdU incorporation, larvae were incubated with 2 mM EdU along with 10 mM HEPES, and the signal was developed using the Click-IT EdU Alexa Fluor 647 kit (Thermo Fischer Scientific). Some of the images have the contrast setting adjusted for visualization purposes.

Karampelias C., Rezanejad H., Rosko M., Duan L., Lu J., Pazzagli L., Bertolino P., Cesta C.E., Liu X., Korbutt G.S, & Andersson O. (2021). Reinforcing one-carbon metabolism via folic acid/Folr1 promotes β-cell differentiation. Nature Communications, 12, 3362.

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