Mice were anesthetized and perfused with phosphate-buffered saline (PBS), and the brain tissues were cut into small pieces and dissociated by enzymatic digestion using the Neural Tissue Dissociation Kit P to obtain the cell suspensions. After the digestion was completed, cells were fixed with 2% PFA for 10 min. Samples were blocked by CD16/CD32 monoclonal antibody for 10 min in FACS buffer (eBioscience, 14016182) followed by incubation with the following primary antibodies for 30 min: a) goat anti-IBA1 pAb (1:50; Abcam, ab5076); b) rabbit anti-NG2 pAb (1:50; Millipore, ab5320). The following secondary antibodies were used: a) Alexa Fluor 488 donkey antigoat IgG (1:200; Invitrogen, A-11055); b) Alexa Fluor 647 goat antirabbit IgG (1:200; Invitrogen, A27040); c) Hoechst (1:1,000; Sigma-Aldrich, 382065). The detection of EdU was performed using BeyoClick™ EdU-647 kit (Beyotime, C0081S) according to the manufacturer’s instructions.
Beyoclick edu 647 kit
Manufactured by Beyotime
Sourced in China
The BeyoClick™ EdU-647 kit is a product designed for the detection and quantification of DNA synthesis in proliferating cells. The kit utilizes the EdU (5-ethynyl-2'-deoxyuridine) labeling method and a fluorescent dye-labeled azide for the visualization of newly synthesized DNA. The core function of this kit is to provide a reliable and efficient tool for cell proliferation studies.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 donkey anti goat igg, by Thermo Fisher Scientific (1 mentions)
Hoechst, by Merck Group (1 mentions)
Goat anti iba1 pab, by Abcam (1 mentions)
Alexa fluor 647 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Facs buffer, by Thermo Fisher Scientific (1 mentions)
Cell counting kit 8 (cck8), by Dojindo Laboratories (1 mentions)
Paraformaldehyde (pfa), by Beyotime (1 mentions)
Fluorescence microscope, by Zeiss (1 mentions)
Triton x 100, by Elabscience (1 mentions)
5 ethynyl 2 deoxyuridine edu, by Thermo Fisher Scientific (1 mentions)
Cytoflex lx, by Beckman Coulter (1 mentions)
10 protocols using beyoclick edu 647 kit
1
Microglial and Oligodendrocyte Progenitor Cells Characterization
2
Cell Proliferation Assay Protocol
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According to the manufacturers' protocols, the cell proliferation assay was performed with the Cell Counting Kit‐8 (Dojindo) and the BeyoClick™ EdU‐647 Kit (Beyotime).
3
Click Reaction of 5-EU in NSN Oocytes
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The click reaction of 5-EU was performed according to the instruction of BeyoClick EdU-647 kit (Beyotime, C0081S) with 5-EU (Click Chemistry Tools, 1261-10) but not the 5-ethynyl-2' -deoxyuridine. In detail, 0.5 mM 5-EU was used to treat the NSN oocytes for 3 h. Then oocytes were picked out for fixation and click reaction. If the transcription of oocytes needs to be inhibited, oocytes were treated with α-amanitin or DMSO for 3 h and then 0.5 mM 5-EU was added for another 3 h. Then oocytes were picked out for click reaction.
4
Intraperitoneal EdU labeling for cell studies
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Mice were intraperitoneally injected with EdU (Beyotime, ST067) (100 μg/mice). The duration of EdU labeling was 2 h for cell experiments in vitro, 1 h for FACS, and 6 h for immunohistochemistry, respectively. Detection was performed using the BeyoClick™ EdU-647 kit (Beyotime, C0081S) according to the manufacturer’s instructions.
5
Measuring DNA Synthesis Dynamics
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EdU incorporation assay was performed to measure the speed of DNA synthesis with a BeyoClick™ EdU-647 kit (Beyotime, Beijing, China).
6
Proliferation Assay with EdU Labeling
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The cells (SW1990 HMGB1 WT & HMGB1 KO) were seeded in 6‐well plates and treated according to grouping conditions. Cells were performed with BeyoClick EdU‐647 Kit (Beyotime, C0081S) according to the manufacturer's protocol. The cells were incubated with 10 µm EdU for 2 h at 37 °C and then postfixed with 4% PFA for 10 min and permeabilized with 0.3% Triton X‐100 in PBS for 15 min, before being incubated with freshly prepared Click‐iT EdU detection cocktail for 30 min at 37 °C. Then incubated with 5 µg mL−1 of DAPI to stain the cell nuclei for 10 min. Images were captured using confocal microscopy.
7
Cell Proliferation Assay with EdU
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Proliferation of the cells was examined using a BeyoClick™ EdU-647 kit (Beyotime). In short, the cells were sorted in 6-well plates. After adherence, the medium was renewed, and each well was loaded with 10 μM EdU solution and the cells were incubated at 37℃ for 2.5 h. Next, the cells were fixed in 4% PFA (Beyotime) for 15 min, permeabilized in 0.3% Triton X-100 (Elabscience Biotechnology Co., Ltd., Wuhan, Hubei, China) for 8 min, and then added with 500 μL Apollo staining buffer in the dark for 40 min. The nuclei were stained with 4', 6-diamidino-2-phenylindole (DAPI) for 10 min. The staining was observed under a fluorescence microscope (Zeiss, Oberkochen, Germany) and quantified using the Image J software.
8
EDU Proliferation Assay in Cultured Cells
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Transfected cells were plated into 96-well plates and cultured for 48 h. An BeyoClick™ EdU-647 kit (Beyotime) was used for EDU assay. Cells were incubated with EDU buffer for 4 h. After that, 4% formaldehyde was used to fix the cells and cell nuclei were stained using DAPI. Lastly, the images were photographed.
9
DNA Replication Analysis via EdU Labeling
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To analyse DNA replication, cells were tagged with 5-ethynyl-2′-deoxyuridine (EdU) and evaluated using the BeyoClickTM EdU-647 Kit (Beyotime Biotechnology, China) according to the manufacturer’s instructions. Cells were incubated in 10 μM EdU buffer for 2 h. Then, cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, then stained with the Click reaction cocktail. Next, DNA was stained with Hoechst 33342 (1 μg /mL in PBS). Finally, flow cytometry was used to identify EdU (BD LSRFortessa, USA).
10
Quantifying Granulocyte Proliferation with EdU
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The proliferation assay was performed with BeyoclickTM EDU-647 kit (Beyotime, Nantong, China) according to the manufacturer’s instructions. Briefly, to detect the percentage of proliferation granulocytes after RAPA treatment, the head kidney granulocytes were collected after 30 min of RAPA treatment, and 20 μg of 5-ethynyl-2′-deoxyuridine (EdU) (Invitrogen, Carlsbad, CA, USA) was added to the granulocyte culture media in vitro. After 1 d, the cells were fixed, permeabilized, and stained for EDU. Then, cells were analyzed using a CytoFLEX LX flow cytometer (Beckman Coulter, Brea, CA, USA).
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