MSCs (100 μL) with (1 × 105)/L density were planted in 96-well plates for induction culture. EdU (KeyGen Biotech. Co., Ltd. Nanjing China) solution was added into the culture wells and the final concentration of EdU was 10 μmol/L. After 37 °C incubation in the 5% CO2 incubator for 24 h MSCs were fixed for 15 min with 4% polyformaldehyde and stained by kFluor488-azide reagent for 15 min at room temperature. Subsequently 0.5 μg/mL Hoechst33342 were employed to re-dye the nuclei for 5 min at room temperature. The images were captured under a fluorescence microscope (Imager M2 Zeiss).
5 ethynyl 2 deoxyuridine (edu)
Manufactured by Keygen Biotech
Sourced in China
EdU is a nucleoside analog that is incorporated into newly synthesized DNA during cellular division. It can be detected by immunofluorescence-based methods, allowing for the visualization and quantification of cell proliferation.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst33342 solution, by Keygen Biotech (2 mentions)
Gelmount containing hoechst 33 342, by Merck Group (2 mentions)
Click it reaction mixture, by Keygen Biotech (2 mentions)
Cell counting kit 8 cck 8 kit, by Dojindo Laboratories (1 mentions)
Bgc 823, by Shanghai Zhong Qiao Xin Zhou Biotechnology (1 mentions)
Mgc 803, by Shanghai Zhong Qiao Xin Zhou Biotechnology (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
N cadherin, by Cell Signaling Technology (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Paraformaldehyde (pfa), by Biosharp (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Polylysine, by Merck Group (1 mentions)
5 ethynyl 2 deoxyuridine (edu), by Merck Group (1 mentions)
Click ittm edu alexa fluor 647 flow cytometry assay kit, by Thermo Fisher Scientific (1 mentions)
Facscanto 2, by BD (1 mentions)
5 ethynyl 2 deoxyuridine (edu), by Zeiss (1 mentions)
Carl microscope, by Zeiss (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
4 6 diamino 2 phenylindole dapi, by Merck Group (1 mentions)
19 protocols using 5 ethynyl 2 deoxyuridine (edu)
1
Proliferation Analysis of MSCs
2
TRIM33 Regulates Gastric Cancer Progression
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The GC cell lines (BGC-823, MGC-803) were purchased from Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd. Human gastric epithelial cells (GES) were purchased from Shanghai Huiying Biological Technology Co., Ltd, and SGC-7901 cells were a gift from Tangshan Yanyi Biotechnology Co., Ltd Roswell Park Memorial Institute (RPMI) medium and fetal bovine serum (FBS) were purchased from HyClone, and F12 Complete Medium from Zhong Qiao Xin Zhou Biotech. siRNA plasmids targeting TRIM33 were synthesized by Life Technologies, and Lipofectamine 2000 was purchased from Invitrogen. Bicinchoninic acid (BCA) and 5-ethynyl-2′-deoxyuridine (EdU) kits were both purchased from Keygen. The rabbit antibody against human TRIM33 (Cat No. 55374-1-AP) was purchased from ProteinTech; rabbit antibodies against human phospho-Smad2 (p-Smad2; #18338), Smad2 (#5339), Smad3 (#9523), Smad4 (#46535), vimentin (#5741), N-Cadherin (#13116), E-Cadherin (#3195), and β-actin (#5125), as well as a horseradish peroxidase-linked goat anti-rabbit antibody (#7074), were purchased from Cell Signaling Technology. Cell Counting Kit-8 (CCK8) kits were purchased from Dojindo, enzyme-linked immunosorbent assays (ELISAs) from Jiangsu Meimian Industrial Co., Ltd, and 24-well plate Transwell chamber systems from Corning.
3
Quantifying mESC Proliferation with EdU
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The mESCs were cultured on confocal plates for 4 days, and then incubated with 10 mM 5-ethynyl-2'-deoxyuridine (EdU; KeyGEN BioTECH, China) for 30 min before fixed with 4% PFA. EdU detection was strictly performed following manufacturer's instructions of keyFluor488 Click-iT EdU imaging detection kit (KeyGEN BioTECH, China) . Experiments were replicated at least three times.
4
Cell Cycle Analysis of RAW264.7 Cells
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After treatment, 2 × 106 RAW264.7 cells were collected and placed in a 37 °C 5% CO2 incubator for 3 h followed by addition of EdU (KeyGEN BioTECH) to each well of the six-well plate at 1/1000 volume for 2 h incubation. Then, cells were collected and stained with DAPI followed by analysis of cell cycle by flow cytometry. The cell cycle distribution was analyzed with FlowJo V10 software.
5
EdU Incorporation Immunofluorescence Assay
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EdU (KeyGEN BioTECH, China) was added to the cell culture medium to a final concentration of 20 μM before incubation at 37 °C for 4 h. The cells were then fixed with 4% paraformaldehyde (Biosharp, USA) for 20 min at room temperature, washed with PBS, and treated with 0.5% Triton X-100 (Sigma, Germany) for 20 min at room temperature. Next, the reaction buffer and KFlour488-azide (KeyGEN BioTECH, China) were mixed and incubated with the cells at room temperature protected from light for 30 min. Lastly, the cells were washed with PBS twice and incubated with Hoechst33342 solution (KeyGEN BioTECH, China) at room temperature in the dark for 30 min.
6
Quantifying DNA Replication via EdU
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EdU incorporation was used to detect DNA replication as described previously.60 (link) Briefly, cells were cultured on coverslips coated with polylysine (20 μg/mL; Sigma-Aldrich) for 36 h in 24-well plates and subsequently incubated with EdU (Keygen Biotech, China) for 12 h, followed by fixation. The staining procEdUre was performed according to the manufacturer’s instructions (Keygen Biotech, China). The coverslips were mounted with Gelmount containing Hoechst 33342 (Sigma-Aldrich). The quantitative results were expressed as the percentage of EdU positive cells out of the total number of cells (as labeled with Hoechst).
7
Cell Proliferation Assay Using EdU
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A549 and H1299 cells were seeded in 96-well plates (2 × 103 cells/well). Next, 100 µl of EdU (50 µM/L, KeyGEN, Nanjing, China) was added to the plates for 2 h of incubation. Cells were fixed with 4% paraformaldehyde in phosphate-buffered saline for 30 min. Cell nuclei were subsequently stained with 4ʹ,6-diamidino-2ʹ-phenylindole and then observed with a fluorescence microscope (Leica, Nikon, Olympus, Zeiss) [20 (link)].
8
EdU Staining for Proliferation Analysis
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EdU staining was performed using kFluor647-EdU cell proliferation assay kit (Keygen Biotech, Nanjing, China). Forty-eight h after transfection, cells were maintained in 10 μM EdU (Keygen Biotech) for 12 h after PBS washing. Then, fixation and penetration of cells were performed, followed by cell incubation in Click-iT reaction mixture (Keygen Biotech). Hoechst 33,342 solution (Keygen Biotech) was used for DNA counter-staining. After mounting, the slices were observed and pictured under a microscope (Olympus, Tokyo, Japan) at 400x magnification.
9
EdU Labeling Assay for DNA Replication Analysis
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EdU labeling assay was performed to examine the rate of DNA replication. In brief, cells were cultured in six-well plate for 24 h followed by an incubation of with 10 μM EdU (KeyGen Biotech, China) in normal culture conditions for 3 h. Cells were harvested and fixed with 4 % paraformaldehyde (PFA). The staining procEdUre was performed according to the manufacturer’s instructions of the Click-iTTM EdU Alexa FluorTM 647 Flow cytometry Assay kit (Thermo). The EdU-positive cells were measured by flow cytometer (BD, FACS Canto II).
10
EdU Incorporation Assay for Glioma Cells
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Cells (75,000) were plated in 24-well plates with round coverslips overnight. 200 µl of EdU (10 µM, KeyGEN Bio TECH, Nanjing, China) were added to each well, and the cells were incubated at 37°C, 2 h. The cells were fixed by adding 4% neutral paraformaldehyde to each well and removed after 30 minutes at room temperature. Then, we added 0.5% TritonX-100 (KenGEN, Nanjing, China) in PBS (200 µl) to each well for 20 min and stained the samples with 200 µl Click-iT reaction mixture (KeyGEN Bio TECH, Nanjing, China) for 30 min under lightproof conditions. Next, DAPI (Beyotime, China) was used to stain the glioma cell nuclei. Round coverslips were observed using Carl Zeiss microscope (Carl Zeiss, Germany) to analyze the proportion of EdU-positive cells.
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