For the EdU tests, BeyoClick EdU-555 Kits manufactured in China by Beyotime were utilized. Cells were grown in a medium that contained 10 μM EdU prior to being fixed with 4% paraformaldehyde and then stained with EdU reaction buffer. The cells were stained with Hoechst so that the DNA could be seen, and the results were analyzed using a fluorescence microscope. Counts were taken of the cells that were positive for EdU.
Beyoclick edu 555 kit
Manufactured by Beyotime
Sourced in China
The BeyoClick EdU-555 kit is a laboratory tool designed for cell proliferation analysis. It enables the detection and quantification of newly synthesized DNA, a key indicator of cell division and proliferation. The kit provides a straightforward and efficient method to label and visualize proliferating cells.
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Lab products found in correlation
Fluorescence microscope, by Olympus (2 mentions)
Tcs sp2, by Leica (1 mentions)
P0126, by Beyotime (1 mentions)
Las x software, by Leica (1 mentions)
Cytexpert 2, by Beckman Coulter (1 mentions)
Bovine serum albumin (bsa), by neoFroxx (1 mentions)
Cytoflex s flow cytometer, by Beckman Coulter (1 mentions)
Dxflex flow cytometer, by Beckman Coulter (1 mentions)
Fluorescent microscope, by Nikon (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Wuhan Servicebio Technology (1 mentions)
Triton x 100, by Beyotime (1 mentions)
Inverted fluorescent microscope, by Olympus (1 mentions)
Hoechst 33258, by Solarbio (1 mentions)
Cytoflex flow cytometer, by Beckman Coulter (1 mentions)
Cck 8, by Beyotime (1 mentions)
Infinite 200 pro microplate reader, by Tecan (1 mentions)
16 protocols using beyoclick edu 555 kit
1
EdU Proliferation Assay Protocol
2
Quantifying Cell Proliferation via EdU Assay
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A total of 4 × 106 cells were seeded on coverslips and subjected to control or TRF paradigm with or without treatment with rapamycin or CQ for 2 consecutive days. Then, BeyoClick™ EdU-488 Kits (C0071S, Beyotime, Shanghai, China) or BeyoClick™ EdU-555 Kits (C0075S, Beyotime, Shanghai, China) were used to detect cell proliferation according to the manufacturer’s protocols. Briefly, 10 μM EdU reagent was added to cells and incubation for 2 h at 37 °C, followed by fixation in 4% paraformaldehyde for 15 min and by permeabilization with 0.3% Triton- × 100 for 15 min at room temperature. Then, cells were washed with PBS and incubated with Click Additive Solution for 30 min at room temperature, protected from light. Further, cells were stained nuclear using Hoechst. Staining was observed under a confocal laser scanning microscope (Leica TCS SP2, Leica, Weztlar, Germany) after sealing with antifade mounting medium (P0126, Beyotime, Shanghai, China). Cell proliferation ability was quantified using Leica LAS X software. Randomly chosen 4 field of views were evaluated for each sample. Three independent experiments were carried out.
3
EdU Assay for Proliferation
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BeyoClick EdU-555 Kits (Beyotime, China) were used for EdU assays. Cells were cultivated in medium containing 10 μM EdU before fixing with 4% paraformaldehyde and subsequent stained with EdU reaction buffer. To visualize the DNA, the cells were stained with Hoechst and observed with fluorescence microscope. The EdU-positive cells were counted.
4
EdU Proliferation Assay for EE Cells
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The effects on the proliferation of EE cells were determined by EdU assay using the BeyoClick™ EdU‐555 Kit (Beyotime Biotechnology) in line with the protocols of manufacturer. Transfected EE cells were seeded in 96 wells and cultured for 2 days. After fixed with 4% paraformaldehyde, cells were added with 5‐ethynyl‐2'‐deoxyuridine (EdU, 10 µM each) and cultivated at 37°C for 2 hours. These cells were subsequently stained using Apollo Dye Solution and DAPI. Finally, the EdU‐positive cells were visualized and analysed through the fluorescence microscope (Olympus).
5
Cellular Proliferation Quantification
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A BeyoClick EdU-555 kit (Beyotime Institute of Biotechnology; cat. no. C0075S) was used to measure the proliferation rate of cells. Firstly, 2×105 cells were inoculated into a 6-well plate and cultured in a cell incubator overnight at 37°C. EdU was added to the medium so that the final concentration of EdU was 20 µM and cells was incubated for another 3 h at 37°C. Subsequently, 4% paraformaldehyde was used to fix the cells for 15 min at indoor temperature and 0.3% Triton X-100 was used for permeabilization. The cells were washed using PBS with 3% BSA (Biofroxx, Germany) twice. The Click Additive solution was then prepared to incubate the cells for 30 min at indoor temperature. The cells were washed again and resuspended in PBS. The fluorescence intensity of 10,000 cells was recorded using a CytoFLEX S flow cytometer (Beckman Coulter, Inc.), before the percentage of EdU-positive cells in each sample was calculated by CytExpert2.3 (Beckman Coulter, Inc.) as follows: FITC positive represents the cells successfully transferred into lentivirus (GFP was detected by FITC). PE-positive represents the EdU-positive cellsand PE-negative represents the EdU-negative cells (EdU was detected by PE).
6
Cell Proliferation Assay with EdU-555
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Cell proliferation was determined using the BeyoClick™ EdU-555 kit (Beyotime Biotechnology) according to the instructions of the manufacturer. Briefly, cells were seeded in 6-well plates at a density of 2.5 × 105 cells/well and incubated at 37°C overnight. Cells were treated with 5-FU or oxaliplatin for 48 h and incubated with 10 μM 5-ethynyl-2′-deoxyuridine (EdU) at 37°C for 2 h. Cells were then harvested by trypsinization, fixed with 4% paraformaldehyde at room temperature (RT) for 15 min, and permeabilized with 0.3% Triton X-100 in PBS at RT for 10 min. Cells were washed, stained with 500 μl of Click Additive Solution at RT for 30 min, and sorted on a DxFLEX flow cytometer (Beckman Coulter, Suzhou, China). EdU-positive cells were analyzed using CytExpert software.
7
KGN Cell Proliferation Assay
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The effects on the proliferation of KGN cells were measured by EdU assay using the BeyoClick™ EdU‐555 Kit (Beyotime Biotechnology, Shanghai, China) in accordance with manufacturer's protocols. Transfected KGN cells were seeded and cultured for 2 day. After fixed with 4% paraformaldehyde, cells were added with 5‐ethynyl‐2'‐deoxyuridine (EdU, 50 μmol\L each) and incubated at 37°C for 2 hours. These cells were subsequently stained with Apollo Dye Solution and DAPI. Finally, the EdU‐positive cells were visualized and analysed under the fluorescence microscope (Olympus, TKY, Japan).
8
EdU-Based Cell Proliferation Assay
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The BeyoClick™ EdU-555 kit (Beyotime, Shanghai, China) manufacturer’s instructions were used as a basis to detect cell proliferation 48 h after transfection. An equal amount of EdU working solution was added to the cell culture medium and cultured for 2 h. The samples were then fixed with 4% paraformaldehyde (Shyuanye, Shanghai, China) and permeabilized with immunostaining permeation buffer containing Triton X-100 (Beyotime, Shanghai, China). The click reaction mixture was added to the wells and incubated for 30 min, and the nuclei were stained using DAPI (Servicebio, Wuhan, China). Finally, images were taken with a fluorescent microscope (Nikon, Tokyo, Japan). Six randomly selected fields of view in each cell well were used to determine the proportion of proliferating cells in each well.
9
Adenovirus-Infected Cell Proliferation
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The proliferation of MG63 and 143B cells was determined using the BeyoClick™ Edu-555 kit (C0075S, Beyotime) according to the manufacturer’s protocol. Both cell lines were seeded at 10,000 cells per well into 24-well plate cell slides after 72-h adenovirus infection. An anti-fluorescence quencher was used to seal the slices, which were then photographed during laser confocal microscopy.
10
Cell Proliferation Assay with EdU
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Cells were seeded at a density of 2 × 104 per well in a 96-well plate for 16 h. Cells were then treated with samples (water and ethanol extracts, different fractions) at 37°C for another 24 h. Next, cells were treated with EdU (20 μM, dissolved in medium) and incubated for 2 h and then fixed with 4% paraformaldehyde for 15 min. Next, cells were permeabilized with 0.5% Triton X-100 at 25°C for 20 min. The EdU staining was followed with the BeyoClick™ EdU-555 Kit (Beyotime, Shanghai, China) and viewed with an inverted fluorescent microscope (Olympus, Tokyo, Japan). Six random fields of vision were captured in each group. Proliferation rate refers to the ratio of the number of EdU stained cells to the number of Hoechst 33342 stained cells. All assays were done three times with triplicate.
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