For colony formation assay, cells were seeded in 6-well plates (4 × 103 cells / well) for two weeks. After fixation, colonies were dyed utilizing 0.1% crystal violet solution (21155722; Biosharp). 5-Ethynyl-2′-deoxyuridine (EdU) staining was conducted with BeyoClick™ EdU-594 cell proliferation assay kit (C0078S; Beyotime, China) following the manufacturer’s instructions. The percentage of EdU-positive cells was calculated.
Beyoclick edu 594 cell proliferation assay kit
Manufactured by Beyotime
Sourced in China
The BeyoClickTM EdU-594 Cell Proliferation Assay Kit is a lab equipment product that enables the detection and quantification of cell proliferation. It utilizes the incorporation of the thymidine analog EdU (5-ethynyl-2'-deoxyuridine) into the DNA of actively dividing cells, which can then be detected using a fluorescent dye.
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5 protocols using beyoclick edu 594 cell proliferation assay kit
1
Colony Formation and Proliferation Assay
2
Evaluation of LTBP2 Silencing on Cell Proliferation
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The activity of HGC-27 and HGC-27/DDPR cells at 0, 24, 48, and 72 h of LTBP2 silencing was detected by referring to the CCK-8 instructions. The proliferation of LTBP2-silenced HGC-27 and HGC-27/DDPR cells was detected by combining the BeyoClick™ EdU-594 Cell Proliferation Assay Kit (C0078S, Beyotime, China) and fluorescence microscopy (DM500, Leica), and the positivity rate was calculated.
In addition, LTBP2-silenced HGC-27 and HGC-27/DDPR cells were inoculated at 300 cells/well into six-well plates. Two millilitres of complete medium was added to each well. Cells were incubated in a constant temperature incubator containing 5% CO2 at 37 °C until the cell clones could be directly observed. The medium was removed, and methanol was added to fix the cells for 30 minutes. The methanol was removed, and the cells were stained with crystal violet for 30 minutes and counted.
In addition, LTBP2-silenced HGC-27 and HGC-27/DDPR cells were inoculated at 300 cells/well into six-well plates. Two millilitres of complete medium was added to each well. Cells were incubated in a constant temperature incubator containing 5% CO2 at 37 °C until the cell clones could be directly observed. The medium was removed, and methanol was added to fix the cells for 30 minutes. The methanol was removed, and the cells were stained with crystal violet for 30 minutes and counted.
3
Quantifying Cell Proliferation with EdU Assay
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The proliferation of the cells was assessed using the BeyoClickTM EdU-594 Cell Proliferation Assay Kit (Beyotime, C0078S). Then, cells were planted in a 24-well plate and incubated until the morphology of the cells returned to normal. Next, 2X EdU working solution was added and incubated. 4% paraformaldehyde was then added. After washing, cells were incubated with a permeabilization solution. Then, cells were incubated with the Click reaction solution out of the light. The nucleus was stained with DAPI (4',6-diamidino-2-phenylindole). A fluorescence microscope was used to photograph and record.
4
Cell Proliferation Assay with EdU Labeling
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Cell proliferation ability was assessed by BeyoClickTM EdU-594 Cell Proliferation Assay Kit (Beyotime, C0078S) following the guidelines. RKO and LoVo cells were planted at a density of 4 × 103 cells per well in 24-well plates. After 24 h of oridonin treatment, the cells were incubated with 10 μM EdU (Beyotime, China) for two hours. Next, the cells were fixed using Immunol Staining Fix Solution (Beyotime, P0098) for 15 min at room temperature. After washing three times with PBS containing 3% BSA, the cells were incubated for 15 min at room temperature in PBS containing 0.3% Triton X-100. The cells were then treated with Click-iT® reaction cocktails at room temperature and out of the dark for 30 min. The fluorescence images were photographed under a fluorescence microscope (Olympus, Japan) after the nuclei were stained by Hoechst 33,342 for 10 min at room temperature. Image J software was utilized to evaluate the percentage of EdU-positive cells.
5
EdU-594 Cell Proliferation Assay
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The BeyoClickTM EdU-594 Cell Proliferation Assay Kit (Beyotime Biotechnology, China) was used to detect DNA synthesis by RA-FLS. The cells were plated in 96-well plates at 1 × 105 cells/well and incubated for 120 min at room temperature with a 20 μM EdU working solution. The cells were fixed with 4% neutral paraformaldehyde and treated with 0.5% Triton X-100 and Click-iT reaction mixture for 0.5 h at room temperature in the dark. The cells were then stained with 5 μg/ml Hoechst 33342 for 15 min at room temperature in the dark, and staining was detected by confocal microscopy (Carl Zeiss, Germany).
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