The proliferation of cells was determined with an EdU-488 Cell Proliferation Kit (Beyotime, China). This analog of thymidine EdU (5-ethynyl-2ʹ-deoxyuridine) can be incorporated into newly synthesized DNA during active DNA synthesis.34 (link) Briefly, after incubation for 24 hrs, cells were cultured with EdU for 2 hrs and then fixed with 4% paraformaldehyde for 15 mins. The cells were permeabilized with 0.1% Triton X-100 for 15 mins and labeled with Azide 488 for the click reaction for 30 mins. Lastly, Hoechst 33342 reagent was added to stain cell nuclei. Specimens were photographed by a fluorescence microscope. The number of EdU-positive cells (green) and total cells (blue) were counted in five randomly chosen fields. EdU-positive ratio was calculated as the ratio of the number of EdU-positive cells divided by the total number of cells.
Protocol detail
Quantifying Cell Proliferation with EdU
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5 ethynyl 2 deoxyuridine
Azide
Cell nuclei
Cell proliferation
Cells
Dna synthesis
Fluorescence microscope
Hoechst 33342
Paraformaldehyde
Stain
Thymidine
Triton x 100
Corresponding Organization : Sichuan University
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Variable analysis
independent variables
- EdU (5-ethynyl-2ʹ-deoxyuridine) incubation time (2 hrs)
- Proliferation of cells
- Ratio of EdU-positive cells to total cells
- Incubation time (24 hrs)
- Paraformaldehyde fixation time (15 mins)
- Triton X-100 permeabilization time (15 mins)
- Azide 488 labeling time (30 mins)
- Hoechst 33342 nuclei staining
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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