The 5-ethynyl-2′-deoxy-uridine (EdU) incorporation assay (Beyotime) was performed to evaluate cell proliferation. Cells were treated with 100 μg/mL of ox-LDL for 24 h and transfected with siCtrl or siFAS for 24 h. In the next step of the study, we transfected the cells with siCtrl or siFAS with miR NC or miR mimic for 24 h, separately. Subsequently, cells were digested and seeded into a 96-well plate at a density of 2X103 cells per well. Cells were cultured with 20 μM of EdU diluent at 37°C with 5% CO2 for 2 h. And then 4% paraformaldehyde was used to fix cells for 15 min. After cells were washed with PBS, 1 mL of permeabilization solution was added to per well and incubated for 15 min at room temperature. Cells were incubated with endogenous peroxidase blocking solution at room temperature for 20 min, and washed with PBS 3 times. 50uL of Click reaction solution was added into each well and incubated for 30 min at room temperature in the dark. After that, cells were washed with PBS 3 times. Streptavidin-HRP working solution was added to the sample, incubated at room temperature for 30 min, and washed with PBS 3 times. After that, 0.1 mL of TMB color developing solution was added, and incubated at room temperature for 30 min. The absorbance was directly measured at 370 nm.
5 ethynyl 2 deoxy uridine edu incorporation assay
Manufactured by Beyotime
Sourced in China
5-ethynyl-2′-deoxy-uridine (EdU) incorporation assay is a technique used to detect and quantify cell proliferation. It is based on the incorporation of a modified nucleoside, EdU, into the DNA of dividing cells. The incorporated EdU can then be detected using a fluorescent azide through a copper-catalyzed click reaction.
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3 protocols using 5 ethynyl 2 deoxy uridine edu incorporation assay
1
EdU Proliferation Assay for ox-LDL and miR Effects
2
Cell Proliferation Assays for MOVAS Cells
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The cell growth was estimated by Cell Counting Kit-8 (CCK8) assay (Beyotime, Shanghai, China) according to the manufacturer’s instructions. MOVAS cells were plated in 96-well plates with 1 × 103 cells per well. After 72 h of treatment, a 10 μL aliquot of CCK-8 reagent was added and incubated for a further 2 h at 37 ℃. Then, optical density (wavelength: 450 nm) was determined by an AMR-100 automatic enzyme label analyzer (Allsheng, Hangzhou, China).
Cell proliferation was measured by 5-ethynyl-2′-deoxyuridine (EdU) incorporation assay (Beyotime, Shanghai, China). After drug treatment, an EdU working solution (10 mM, with green fluorescence) was added to the medium and then incubated for 2 h. After incubation, the cells were fixed with 4% PFA, washed with PBS, permeabilized with 0.3% Triton X-100, added to a freshly prepared Click reaction solution, and the cells were then stained with Hoechst 33342.
Cell proliferation was measured by 5-ethynyl-2′-deoxyuridine (EdU) incorporation assay (Beyotime, Shanghai, China). After drug treatment, an EdU working solution (10 mM, with green fluorescence) was added to the medium and then incubated for 2 h. After incubation, the cells were fixed with 4% PFA, washed with PBS, permeabilized with 0.3% Triton X-100, added to a freshly prepared Click reaction solution, and the cells were then stained with Hoechst 33342.
3
Evaluating Gastric Cancer Cell Proliferation
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Cell proliferative ability of gastric cancer cells was evaluated by 5′Ethynyl-2′-deoxyuridine (EdU) incorporation assay (Beyotime). The gastric cancer cells including SGC-7901 and MKN-28 with different interventions were seeded at 5 x 104 cells/well, after 24 h culture, the cells were incubated with EdU solution for 2 h. After that, the cells were Apollo reaction cocktail and subsequently stained with 4′, 6-diamidino-2-phenylindole. The EdU-positive cells were evaluated using the fluorescent microscopy. The percentage of EdU-positive cells was calculated by dividing the number of EdU-positive cells by the number of DAPI-stained cells.
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