Edu incorporation assays were used to determine cell proliferative abilities. Cells in logarithmic growth were taken and cultured in 6-well plates. Edu labeling solution (20 μM, Beyotime, Shanghai, China) was added to the 6-well plates 48 h after transfection and then incubated for 2 h at 37°C and 5% CO2. The cells were stained with the Click Additive Solution (Beyotime, Shanghai, China) and Hoechst 33342, and an anti-Edu working solution according to the manufacturer’s instructions. After this, cells were treated with 4% paraformaldehyde and PBS containing 0.3% Triton X-100. The percentage of Edu-positive cells was finally measured using fluorescence microscopy analysis.
Click additive solution
Manufactured by Beyotime
Sourced in China
The Click Additive Solution is a specialized laboratory equipment product. It serves as a core functional component for various applications in the scientific and research domains. The product enables specific technical processes, but a detailed description of its intended use is not available.
Automatically generated - may contain errors
Lab products found in correlation
5 ethynyl 2 deoxyuridine (edu), by Beyotime (2 mentions)
Beyoclick edu kit, by Beyotime (2 mentions)
Hoechst 33342, by Beyotime (2 mentions)
Beyoclick edu cell proliferation kit with alexa fluor 555, by Beyotime (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Triton x 100, by Beyotime (1 mentions)
Paraformaldehyde (pfa), by Wuhan Servicebio Technology (1 mentions)
5 ethynyl 2 deoxyuridine edu, by Thermo Fisher Scientific (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
Fv1000, by Olympus (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Hoechst, by Beyotime (1 mentions)
Dmi8 fluorescence microscope, by Leica (1 mentions)
8 protocols using click additive solution
1
Edu Incorporation Assay for Cell Proliferation
2
Analysis of Cell Proliferation via EdU Labeling
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Cells were seeded on glass coverslips in 24 well plates. After treatment, cells were incubated with Edu (Beyotime Biotechnology, Shanghai, China) for 2 h at 37 °C, and fixed in 4% paraformaldehyde. After permeabilization with 0.5% Triton-X, the cells were reacted with click additive solution (Beyotime Biotechnology) for 30 min. Subsequently, the DNA contents of the cells were stained with Hoechst 33342 for 10 min and visualized under a fluorescence microscope. Edu positive cells were quantified from three randomly selected fields in each well, and each experiment was repeated for three times.
3
Evaluating Cell Proliferation via EdU Staining
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To assess DNA synthesis, the HCT-116, RKO or HT-29 CRC cells were subjected to EdU staining using the BeyoClick™ EdU Cell Proliferation kit with Alexa Fluor 555 (Beyotime Institute of Biotechnology), according to the manufacturer's instructions. A total of 5×105 HCT-116 and RKO cells transfected with a negative control siRNA or a siRNA against ZNF169, or a total of 4×105 HT-29 cells infected with an empty control or ZNF169 overexpression lentivirus, were seeded onto coverslips in six-well plates. After 24 h, the cells were incubated with EdU reagent at 37°C for 4–6 h. The cells on the coverslips were then washed with PBS, fixed with 4% paraformaldehyde (Wuhan Servicebio Technology Co., Ltd.) for 15 min at room temperature, treated with 0.5% Triton X-100 (Beyotime Institute of Biotechnology) for 15 min at room temperature, and finally stained with Click Additive Solution (Beyotime Institute of Biotechnology) for 30 min at room temperature and DAPI (Beyotime Institute of Biotechnology) for 15 min at room temperature in the dark. Images of the EdU-positive cells were captured under a microscope and quantified by Photoshop.
4
EdU Labeling of Proliferating Cells
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Cells were exposed to 10 µM of 5-ethynyl-2′-deoxyuridine (EdU; Thermo Fisher Scientific) for 1 h at 37 °C. The cells were fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and reacted with Click Additive Solution (Beyotime, Shanghai, China) for 30 min in the dark. Nuclei were stained with Hoechst 33,342 (Beyotime). Proliferating cells were visualized under a fluorescence microscope.
5
Cell Proliferation Assay using EdU
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EdU staining was performed to analyze cell proliferation using the BeyoClick™ EdU kit (Beyotime Institute of Biotechnology). Briefly, treated HemECs (as described in ‘BLM and PYM treatment’ and ‘740 Y-P treatment’ subsections) were seeded in 6-well plates (3x105 cells/well) and incubated overnight at 37˚C. EdU (10 µM) was added to each well and cells were incubated for 2 h at 25˚C. HemECs were fixed in 4% paraformaldehyde at room temperature, permeabilized by 0.3% Triton X-100 and stained with Click Additive Solution (Beyotime Institute of Biotechnology) according to the manufacturer's protocol. Cells were imaged using a fluorescence microscope (Olympus Corp.) under a magnification of x200. The proportion of EdU-positive cells was calculated as follows: (Red fluorescent spot number/blue fluorescence spot number) x100.
6
EdU Incorporation Assay for Proliferation
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Cells were inoculated into 24-well culture plates for 24 h. A concentration of 50 μM 5-ethynyl-2’-deoxyuridine (EDU; C0075S-1, Beyotime, Biotechnology) was added and incubated for 1.5 h. Cells were then washed with PBS, fixed with 4% paraformaldehyde for 15 min, and permeabilized with 0.5% Triton X-100 for 10 min. Subsequently, cells were treated with 200 μL Click Additive Solution (C0075S-5, Beyotime, Biotechnology) at room temperature and protected from light for 30 min. Cells were then stained with 200 μL of 1× Hoechst 33342 (C0075S-6, Beyotime Biotechnology) at room temperature for 30 min under a laser scanning confocal focusing microscope (FV-1000; Olympus, Tokyo, Japan). Five areas of positive cells were randomly counted.
7
Cell Proliferation Assay with BI 2536
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Cells were seeded in a 12-well plate. After the treatment with indicated drugs (DMSO, 0.5 μM BI 2536) for three days, EdU (Beyotime Biotec) was added at a working concentration of 10μM for 2 hours. The cells were washed with PBS, fixed with 4% formaldehyde for 30 min, permeabilized with 0.25% Triton X-100 (Sigma), and incubated with Click Additive Solution (Beyotime Biotec) according to the manufacturer’s instructions, following by staining with Hoechst (Beyotime Biotec). After the staining, the cells were observed using a fluorescent microscope (Olympus).
8
EdU Staining for Cell Proliferation
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Cell proliferation was assessed using an EdU staining using the BeyoClick EdU kit (Beyotime Institute of Biotechnology), according to the manufacturer's protocol. Briefly, cells were treated with PBS, CDDP, MG132, or CDDP + MG132 for 48 h and were then incubated with 10 µM EdU at 37˚C for an additional 2 h. Following washing with PBS, cells were fixed with 4% polyformaldehyde at RT, permeabilized with 0.3 Triton X-100 followed by incubation with Click additive solution (Beyotime Institute of Biotechnology) at RT for 30 min in the dark. After staining with EdU, cells were counterstained with Hoechst 33342 for 10 min at RT and observed under a Leica fluorescence microscope DMi8 (Leica Biosystems; magnification, x200).
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