Cell Counting Kit-8

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	- Cells were incubated for 2 h at 37 °C after addition of CCK-8 reagent

Protocol tips
- Cells were incubated for 2 h at 37 °C after addition of CCK-8 reagent

Publication protocol

The proliferation of the HUVECs was determined using a CCK-8 assay (Dojindo Molecular Technologies, Inc.) and a 5-ethynyl-2’-deoxyuridine (EdU) assay using an EdU assay kit (Ribobio, Guangzhou, China), as previously described[14, 15]. HUVECs were cultured in 96-well plates at a density of 5×103 cells per well and transfected with the miR-497 mimic, miR-497 inhibitor, or their respective control RNA for 48 h. For the CCK-8 assay, 10 μL of the CCK-8 reagent was added to each well for 2 hours. Then, the absorbance at 450 nm was measured using an Enzyme mark instrument. For the EdU assay, 50 μM EdU was added to the cells, and the cells were incubated for 2 h at 37°C. The cells were then fixed with 4% paraformaldehyde for 15 min at room temperature and exposed to 0.5% Triton X-100 for 20 min. After 3 washes with PBS, the cells were stained with 100 μL of Apollo Dye Solution for 30 min. The nucleic acids in all of the cells were stained with DAPI. Images were taken using a fluorescence microscope (Axio Observer Z1, Carl Zeiss. Inc). All experiments were performed in triplicate.


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