The proliferation of the control and overexpression groups at 0 h and 12 h was observed under a microscope (Leica, Heidelberg, Germany). Cell Counting Kit-8 (CCK8) and 5-ethynyl-2-deoxyuridine (EdU) assays were used to analyze cell proliferation, which were performed as previously described [43 (link)]. Briefly, control and transfected cells were incubated with 10% CCK8 (Beyotime Biotechnology, Shanghai, China) at 37 °C for 1 h in the dark, and the absorbance was measured at 450 nm to determine the proliferation ability. For EdU staining, cells were incubated with 50 mM EdU (Ribobio, Guangzhou, China) at 37 °C for 2 h. EdU-positive cells were analyzed in the different treatment groups with Image J software.
50 mm edu
Manufactured by RiboBio
Sourced in China
50 mM EdU is a concentrated solution of the chemical compound 5-ethynyl-2'-deoxyuridine, a thymidine analogue used in various cellular and molecular biology applications. This product provides a consistent concentration for researchers to work with.
Automatically generated - may contain errors
Lab products found in correlation
Cck 8, by Beyotime (1 mentions)
Fluorescence microscope, by Zeiss (1 mentions)
5 ethynyl 2 deoxyuridine edu assay kit, by RiboBio (1 mentions)
Epoch microplate spectrophotometer, by Agilent Technologies (1 mentions)
Apollo reaction cocktail, by RiboBio (1 mentions)
Cck 8 cell proliferation kit, by Dojindo Laboratories (1 mentions)
Apollo staining solution, by RiboBio (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Roche (1 mentions)
3 protocols using 50 mm edu
1
Cell Proliferation Assays Protocol
2
Measurement of Cell Proliferation
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Cell proliferation was measured using the CCK-8 cell proliferation kit (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) and 5-ethynyl-2′deoxyuridine (EdU) assay kit (Guangzhou Ribobio Co., Ltd., Guangzhou, China), respectively. For the CCK-8 assay, siRNA-PYCR1 or control siRNA-transfected cells were cultured in a 96-well plate at a density of 3,000 cells per well. CCK-8 solution (20 µl) was added to each well after 0, 24, 48, 72 and 96 h. Cells were incubated at 37°C for 2 h, and the absorbance of samples was recorded at 450 nm using an epoch microplate spectrophotometer (BioTek Instruments, Inc., Winooski, VT, USA). All the experiments were performed in triplicate.
For the EdU incorporation assay, dissociated cells were exposed to 50 mM EdU (Guangzhou RiboBio Co., Ltd.) for 2 h at 37°C. Following fixation with 4% formaldehyde for 15 min and permeabilization with 0.5% Triton X-100 for 10 min at room temperature, the cells were incubated with 1X Apollo reaction cocktail (Guangzhou RiboBio Co., Ltd.), 100 µl/well for 30 min. Then, the cells were stained with Hoechst 33342 for 30 min at room temperature and visualized in 3 fields of view/well under a fluorescence microscope (Carl Zeiss). The EdU incorporation rate was expressed as the ratio of EdU-positive cells to total Hoechst 33342-positive cells.
For the EdU incorporation assay, dissociated cells were exposed to 50 mM EdU (Guangzhou RiboBio Co., Ltd.) for 2 h at 37°C. Following fixation with 4% formaldehyde for 15 min and permeabilization with 0.5% Triton X-100 for 10 min at room temperature, the cells were incubated with 1X Apollo reaction cocktail (Guangzhou RiboBio Co., Ltd.), 100 µl/well for 30 min. Then, the cells were stained with Hoechst 33342 for 30 min at room temperature and visualized in 3 fields of view/well under a fluorescence microscope (Carl Zeiss). The EdU incorporation rate was expressed as the ratio of EdU-positive cells to total Hoechst 33342-positive cells.
3
Edu Labeling and DAPI Staining
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The cells were incubated with 50 mM Edu (RiboBio, Guangzhou, China) for 12 h and then fixed with 4% paraformaldehyde at 25 °C for 30 min. Next, the cells were incubated in PBS supplemented with 0.3% triton x-100 on a decoloring shaker for 10 min. The cells underwent further incubation in Apollo staining solution (RiboBio, China) for 20 min, then in NaCl/Pi (3 times, 10 min for each, room temperature), and finally with 4′-6-diamidino-2-phenylindole (DAPI, 1: 2500; Roche, Mannheim, Germany) at room temperature for 10 min.
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