Edu 488 cell proliferation kit

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.

Overall cell proliferation was measured by MTT-based cell viability assay. Put simply, cells were cultured in 96-well plates, and corresponding treatment was performed when the cell density reached about 50 %. After 24 h, 5 mg/ml of MTT (BioFroxx Cat #1334GR005) solution was added and the incubation was continued for 4 h at 37 °C. Finally, discard the supernatant fractions, add 200 μl DMSO to each well, incubated for 10 min at room temperature on a shaker, and detect the wavelength at 490 nm. For individual cell proliferation, Edu-488 cell proliferation kit was used to detect (Beyotime Biotechnology Cat# C0071S). HepG2 and Huh7 cells were inoculated into 96-well plates. Then labeled by 37 °C pre-warmed EdU 2 h prior to sample collection. After labeling, the cells were fixed with 4 % paraformaldehyde for 10 min and permeabilized with 0.3 % Triton X-100. Then assayed the fluorescence intensity according to the steps in the instructions.