EDU Proliferation Assay Protocol

The EDU assay was performed as described previously [22 (link)]. In short, cells (1 × 10⁵) were seeded in 24-well plates and cultured for 20 h. After incubation with EDU reagent for 2 h, cells were fixed with 4% paraformaldehyde (solarbio) and permeabilized with 0.5% Triton X-100 (solarbio). Sequentially, cells were counterstained following the manufacturer’s instructions of the EDU assay kit (Beyotime, Biotechnology, China). The images were captured using a laser scanning confocal microscope (Olympus) and then the EDU-positive cells percentage could be calculated.

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Hu J., Zhang G., Wang Y., Xu K., Chen L., Luo G., Xu J., Li H., Pei D., Zhao X., Guo Z., Li X., Zong S., Jiang Y, & Jing Z. (2023). CircGNB1 facilitates the malignant phenotype of GSCs by regulating miR-515-5p/miR-582-3p-XPR1 axis. Cancer Cell International, 23, 132.

Publication 2023

paraformaldehyde Triton X-100 EDU assay kit laser scanning confocal microscope

Assay Cells Laser scanning confocal microscope Paraformaldehyde Triton x 100

Corresponding Organization : Tongji University

Other organizations : First Hospital of China Medical University, China Medical University

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Variable analysis

independent variables

EDU reagent incubation duration (2 h)

dependent variables

Percentage of EDU-positive cells

control variables

Cell seeding density (1 × 10^5 cells)
Culture duration (20 h)
Fixation with 4% paraformaldehyde
Permeabilization with 0.5% Triton X-100

controls

No positive or negative controls were explicitly mentioned.

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