Quantifying Cell Proliferation with EdU Labeling

5-Ethynyl-2′-deoxyuridine (EdU) (Click-iT EdU Alexa Fluor 488 Imaging Kit, Invitrogen) is a nucleoside analog of thymidine. It is incorporated into DNA during active DNA synthesis. Cell proliferation was determined by the EdU incorporation assay, as previously described10 (link). Briefly, 100 μL of 2× working solution of EdU was added to the wells with FNSCs in the 96-well plate during the exposure time for 6 hours with different sevoflurane concentrations. After incubation, the cells were fixed with 4% paraformaldehyde for 15 min and then 0.2% Triton X-100 for 20 min at room temperature. The cells in each well were washed twice with 3% BSA between every two steps. Then, 100 μL of Click-iT reaction cocktail was added to each well for 30 min and protected from light. The cells were blocked with 3% BSA for 1 h and then incubated with the anti-nestin antibody (Millipore; 1:500 dilution) overnight at 4 °C. After washing with the 3% BSA, the cells were incubated with the secondary antibody Alexa Fluor 555 for 1 h and then with the 1× Hoechst 33342 solution for 10 min in the dark at room temperature. Each well was washed twice with PBS, and the images were obtained using an Olympus IX-70 inverted fluorescence microscope(Olympus America Inc, Center Valley, PA).