Edu cell proliferation kit with alexa fluor 488

After removed the cells with 0.25% trypsin (GIBCO), the cell suspension was rinsed by pre-cooled PBS and then gently re-suspended with 70% ethanol at 4 °C for 12 h. The fixed cells were rinsed with PBS and incubated with propidium iodide containing RNase A at 37 °C for 30 min (Beyotime, C1052). The single cells were screened before flow analysis (Cytomic FC 500 MCL, Beckman Coulter, USA). Data was obtained using CXP software v2.2 (Beckman Coulter, USA), and set the parameter to FL3-620nm BP. 10,000 events were collected per sample manually determined the percentage of G1, G2 and S phases. Data in different groups were imported in Excel software (Microsoft) and generated the graph of relative cell population.
The S phase cells were detected by EdU Cell Proliferation Kit with Alexa Fluor 488 (Beyotime, C0071S). The cells on coverslip were treated with pre-warmed EdU (20 µM) for 2 h, and then fixed by 4 % paraformaldehyde in PBS for 15 min and permeabilized with 0.25% Triton X-100 in PBS for 10 min. Each process followed by PBS washing for 3 times and the cells were incubated in click additive solution (Click Reaction Buffer, CuSO₄ and Azide 488) at room temperature for 30 min. The followed steps of staining the antibodies and nucleus were same as the immunofluorescence assay.

The EdU assay was conducted using EdU Cell Proliferation Kit with Alexa Fluor488 (Beyotime, Shanghai, China) according to the manufacturer’s protocol. Briefly, A549 and H1299 cells were plated onto the 12-well plates and incubated with apatinib (0, 2, 5, 10 μM) for 48 h. After incubation of 10 μM EdU for 2 h, cells were fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.3% Triton X-100 for 20 min. Then, the incorporated EdU was visualized by means of a click reaction using Alexa Fluor 488 azide for 30 min and the nuclear DNA was stained with 4, 6-diamino-2-phenyl indole (DAPI, Beyotime) for 10 min. Finally, the proliferative cells were observed using a fluorescence microscope (Nikon) and the percentage of EdU positive cells was assessed using the ImageJ software.

The EdU Cell Proliferation Kit with Alexa Fluor 488 was purchased from Beyotime (China). All steps were conducted in accordance with the manufacturer's instructions. In brief, pancreatic cancer cells were treated with varying concentrations of linderalactone, and then, EdU was added and incubated with the cells for one-tenth of the cell doubling time (BXPC-3, 5 h; CFPAC-1, 3 h). The cells were incubated with Hoechst stain for 20 min to visualize the nucleus and then examined under a fluorescence microscope (Nikon, Japan).