Click it plus edu imaging kit

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Click-iT Plus EdU Imaging Kit is a tool for the detection and visualization of DNA synthesis in proliferating cells. It utilizes a modified nucleoside, EdU (5-ethynyl-2'-deoxyuridine), which is incorporated into newly synthesized DNA. The incorporated EdU can then be detected and visualized through a copper-catalyzed click reaction with a fluorescent azide.

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64 protocols using click it plus edu imaging kit

Immunofluorescence and EdU Labeling in Mouse Brain

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For immunofluorescence, mice were perfused with 50 ml of PBS followed by 50 ml of 4% paraformaldehyde. The brains were harvested and fixed for 24 hrs in 4% paraformaldehyde. The samples were transferred to 30% sucrose in PBS, and frozen at OCT in −80°C until sectioning. at 40 μM sections were prepared using a Leica CM1950 cryostat. For staining of microglia, tissues were stained with goat anti-Iba-1 (Thermo), and Cy5-conjugated anti-goat secondary antibody (Jackson ImmunoResearch). Slides were imaged using a Zeiss Axio Observer fluorescence microscope with a 20X objective. The Zeiss Zen Blue software (Zeiss) was used to process the images.
For in vivo EdU labeling, mice were injected i.p. with 6 doses of EdU (100 mg/kg) over 2 days. EdU was detected by the Click-iT Plus EdU Imaging Kit (Invitrogen) according to the manufacturer’s instructions. After EdU detection, the sections were stained with anti-NeuN rabbit polyclonal ab (A60) (Millipore) and anti-rabbit Rhodamine secondary ab (Jackson). Slides were imaged using a Zeiss Axio Observer fluorescence microscope with a 10X objective. Images were processed by the Zeiss Zen Blue software (Zeiss).

Zhang Y., Fung I.T., Sankar P., Chen X., Robison L.S., Ye L., D’Souza S.S., Salinero A.E., Kuentzel M.L., Chittur S.V., Zhang W., Zuloaga K.L, & Yang Q. (2020). Depletion of NK cells improves cognitive function in the Alzheimer’s Disease mouse model. Journal of immunology (Baltimore, Md. : 1950), 205(2), 502-510.

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EdU Labeling and Immunostaining of Embryonic Gut

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Embryonic day (E)12.5 or E14.5 pregnant females were injected intraperitoneally with EdU (50 μg/g body weight). Two hours after injection, dissection of gut was followed by fixation and permeabilization in the same fashion as whole-mount immunostaining. The preparations were washed twice for 3 min with 3% BSA in PBS at room temperature. For EdU assays (Click_-iT Plus EdU Imaging kit, Invitrogen), the reaction cocktail (reaction buffer, CuSO₄, Alexa Flour 594 azide and buffer additive as per manufacture’s protocol) was added for 30 min followed by rinsing twice for 3 min with 3% BSA in PBS in the dark at the room temperature. After EdU labeling, whole-mount immunostaining was performed as described above.

Okamoto M., Uesaka T., Ito K, & Enomoto H. (2021). Increased RET Activity Coupled with a Reduction in the RET Gene Dosage Causes Intestinal Aganglionosis in Mice. eNeuro, 8(3), ENEURO.0534-20.2021.

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Generation of EdU-labeled HSV-1 Virus

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HSV-1 recombinant virus T1012G was previously described¹⁵. EdU genome-labeled T1012G (T1012G^EdU) was generated as follows. Vero(CCL-81) cells were infected with 0.1 MOI T1012G and incubated until a complete cytopathic effect developed. EdU was added to the cells at 2 h and 24 h post-infection to a final concentration of 10 μM. EdU was from Click-iT Plus EdU imaging kit purchased from Invitrogen. The cell medium was harvested at approximately 72 h post-infection, and cell debris was removed by low-speed centrifugation. The virus-containing cells were collected, aliquoted and stored at − 80 °C for further titration analysis.

Ren Y., Chen M., Wu G., Ji D., Zhou G.G., Ren P.G, & Fu W. (2021). High accumulation of Mx2 renders limited multiplication of oncolytic herpes simplex virus-1 in human tumor cells. Scientific Reports, 11, 21227.

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Quantifying Cell Proliferation via EdU

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The EdU assay was performed using the Click-iT® Plus EdU Imaging Kit (Invitrogen) according to the manufacturer's instructions. The percentage of EdU⁺ cells was determined by counting an average of 500-1,000 cells per field from 3 randomly selected sample regions using ImageJ software.

Duan G., Song Z., Qi M., Bai X., Wang J., Zhang Y., Zou X., Guo Q, & Wan P. (2018). Increased Autophagy Levels Mediate Cisplatin Resistance in Cisplatin-Resistant Cells While Also Rendering Them Vulnerable to Autophagy Induction. BioMed Research International, 2018, 1736738.

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Visualizing Proliferating Cells in Zebrafish

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The embryos were treated with 2 mM 5-ethynyl-2-deoxyuridine (EdU) for 20 min at 4 °C and then washed 3 times with egg water. The embryos were transferred to egg water in an incubator at 28.5 °C. After 4 h, the embryos were fixed in 4% paraformaldehyde overnight, dehydrated in a methanol gradient overnight and rehydrated. Zebrafish embryos were used for frozen sectioning, and then the Click-iT Plus EdU Imaging Kit (Invitrogen, C10640) was used to process the sections for 30 min. The reaction was terminated using PBS with 0.1% Tween. N ≥ 6, the experiment was repeated at least three times.

Zhang R., Sun J., Xie Y., Zhu W., Tao M., Chen Y., Xie W., Bade R., Jiang S., Liu X., Shao G., Pan W., Zhou C, & Jia X. (2024). Mutant kri1l causes abnormal retinal development via cell cycle arrest and apoptosis induction. Cell Death Discovery, 10, 251.

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Swine Intestinal Organoid Proliferation

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Single-cell suspensions from swine intestinal organoids were generated by TrypLE and counted. About 3,000 cells per replicate were plated in Matrigel and incubated in the PIO medium described above. At the indicated time points, 700 uM resazurin (Sigma) in PBS was added 10% by volume to each treatment well. Cells were incubated for 5 h in 37°C, 5% CO₂ incubator, and then fluorescence was measured on BioTek Epoch Microplate Spectrophotometer (BioTek).
The proliferation of organoids was also evaluated by Click-iT Plus EdU Imaging Kit (Invitrogen). In brief, organoid cells were incubated with EdU for 6 h fixed in 4% paraformaldehyde in DPBS and incubated for 15 min at room temperature. After washing 3 times with DPBS, the organoids were permeabilized and blocked with blocking buffer (0.1% Triton-X 100 and 3% BSA in DPBS) for 20 min at room temperature. Click-iT Plus reaction cocktail was added and incubated with cells for 30 min. The cells were counterstained with Hoechst for 10 min. Images were taken by Olympus IX81 fluorescence microscope.

Zhang M., Lv L., Cai H., Li Y., Gao F., Yu L., Jiang Y., Tong W., Li L., Li G., Tong G, & Liu C. (2022). Long-Term Expansion of Porcine Intestinal Organoids Serves as an in vitro Model for Swine Enteric Coronavirus Infection. Frontiers in Microbiology, 13, 865336.

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NSC Proliferation Assay with Particle Treatments

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The NSCs were plated at a 1:4 split ratio on 8-well CC2 chamberslides (Nunc, ThermoFisher, Waltham, MA, USA) coated with laminin (Invitrogen, ThermoFisher, Waltham, MA, USA) overnight. The next day, the medium was replaced with fresh medium containing 10 μg/ml PS-MPs or NPs, and cells were incubated for 18 h. For EdU incorporation, cells were incubated in N5 medium containing 5 μM EdU for 1 h. The EdU detection assay was performed following manufacturer's protocol of the Click-iT Plus EdU Imaging Kit (Invitrogen, ThermoFisher, Waltham, MA, USA). Briefly, cells were fixed in 4 % paraformaldehyde for 15 min and then, permeabilized with 0.5 % Triton X-100 for 20 min. Cells were incubated in EdU detection cocktail for 30 min, followed by 4′,6-diamidino-2-phenylindole (DAPI) staining (1:1000 diluted in phosphate buffered saline (PBS)) for 30 min. Between different solutions, 3 % bovine serum albumin (Sigma, St. Louis, MO, USA) in PBS was used for washing. After imaging with the fluorescence microscope (Olympus, Tokyo, Japan), EdU-positive cells and DAPI-stained cells were counted using a cell count macro in iSolution software (IMT i-Solution Inc., Vancouver, BC, Canada).

Park K.Y., Kim M.S, & Oh N. (2024). Cytotoxicity of amine-modified polystyrene MPs and NPs on neural stem cells cultured from mouse subventricular zone. Heliyon, 10(10), e30518.

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Detection of DNA Replication using EdU

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To detect DNA replication using the thymidine analogue EdU, either the Click-iT EdU imaging kit or the Click-iT Plus EdU imaging kit (Invitrogen) was used [74 ]. Adult flies were maintained on medium containing 0.2 mM EdU (ThermoFisher) from eclosion until dissection at 6 days. The EdU-containing medium was prepared by mixing standard cornmeal agar medium with a 10 mM stock solution (diluted in PBS per the manufacturer’s instruction). To detect EdU incorporation, dissected AGs were fixed in paraformaldehyde and processed as they were for immunohistochemistry. The Click-iT EdU reaction mix was prepared following the manufacturer’s instructions using a 1:400 dilution of a 2 mM stock of azide-fluor 555 (Sigma-Aldrich) dissolved in DMSO (this preparation was not required for the Click-iT Plus EdU kit). To label EdU-containing DNA in the sample, 200 μl of the reaction mix was added to the vials and left to incubate away from light for 30 minutes at 20°C. Glands were washed three times in 200 μl PBST and then resuspended in 200 μl PBS before mounting on coverslips using DAPI-containing mounting medium (Vectorshield; Vector Laboratories).

Leiblich A., Hellberg J.E., Sekar A., Gandy C., Mendes C.C., Redhai S., Mason J., Wainwright M., Marie P., Goberdhan D.C., Hamdy F.C, & Wilson C. (2019). Mating induces switch from hormone-dependent to hormone-independent steroid receptor–mediated growth in Drosophila secondary cells. PLoS Biology, 17(10), e3000145.

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Cell Proliferation Assay in Mice

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Mice were injected with EdU (10 mg/kg in PBS) intraperitoneally 1 h prior to tissue collection. Cells were treated with 10 μg/ml for 1h before fixation in 4% formaldehyde. Click-iT Plus EdU Imaging Kit (Invitrogen, Grand Island, NY) with AlexaFluor 488 (Cat.#C10637) or 647 (Cat.#C10639) was used according to the manufacturer’s recommendations. Slides were mounted in ProLong™ gold antifade medium with DAPI (ThermoFisher Scientific, Cat.#p36931). Percentage of cells with EdU+ nuclei and abnormal nuclei were determined by counting under the microscope in randomly selected three areas (at least 200 cells in each area).

Fedtsova N., Komarova E.A., Greene K.F., Novototskaya L.R., Molodtsov I., Brackett C.M., Strom E., Gleiberman A.S., Shakhov A.N, & Gudkov A.V. (2021). Resistance of bone marrow stroma to genotoxic preconditioning is determined by p53. Cell Death & Disease, 12(6), 545.

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Quantifying Intestinal Stem Cell Renewal

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5-ethynyl-2′-deoxyuridine (EdU) staining was performed following the manufacturer’s protocol (Click-iT Plus EdU imaging kit, Invitrogen). Briefly, crypts or single cells were cultured on ibidi µ-Slides coated with Matrigel, explained above. After overnight in culture, cells were incubated with EdU (10 µM) for 2 h, washed with PBS, and cultured with ENR_CV-medium. Samples were fixed with 3.7% formaldehyde (Electron Microscopy Sciences) immediately (chase 0 h) or after 24, 48, 72 and 96 h (chase 24 h, 48 h, 72 h and 96 h) after the EdU pulse. After fixation, samples were permeabilized with 0.5% Triton X-100 in PBS for 20 min and EdU was detected using Click-iT Plus EdU Alexa Fluor 488 imaging kit. Subsequently, the samples were washed with 3% BSA in PBS and incubated with primary antibodies anti-Ki67 (1:100) and anti-ZO-1 (1:200) overnight at 4 °C, followed by incubation with the secondary antibodies, Alexa Fluor 647 and Alexa Fluor 568, for 1 h at RT. Nuclei were stained with Hoechst 33342 (10 µg mL⁻¹, Click-iT, Invitrogen) for 30 min at RT.

Altay G., Larrañaga E., Tosi S., Barriga F.M., Batlle E., Fernández-Majada V, & Martínez E. (2019). Self-organized intestinal epithelial monolayers in crypt and villus-like domains show effective barrier function. Scientific Reports, 9, 10140.

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