Beyoclick edu 647 cell proliferation kit

EdU assay was implemented in ACHN and 786-O cells according to manufacturer’s protocol by use of the BeyoClick™ EdU-647 Cell Proliferation Kit (Beyotime, China). After transfection, cells (1 × 10⁵ per well) were seeded into 6-well plates. 24 h later, 10μM EdU medium was added into cells for 2 h. Next, 4% paraformaldehyde (PFA) was added for 15-min fixing. After three-time rinse by washing buffer (3% BSA), cells were washed by permeabilization buffer (0.3% Triton X-100) for 15 min. After one-time rinse by washing buffer, Click additive reaction system was added to label the proliferated cells and Hoechst 33342 was added for cell counting. Finally, cells were visualized using fluorescence microscope (Olympus, Japan). Each independent experiment was carried out in duplicate.

For the cell viability assay, the transfected DC-CIK cells or T cells and cocultured leukemia cells were plated into a 96-well plate and incubated for 24, 48, and 72 h. Then, cells were incubated in 10% CCK-8 working solution (5 mg/mL, Beyotime Institute of Biotechnology, Shanghai, China) in the dark for 1 h. The absorbance at 450 nm was recorded using a microplate reader (BioTek Instruments, Winooski, VT, USA). The evaluation of DC-CIK cells or T cells and cocultured leukemia cell proliferation by EdU staining was finished using the BeyoClick EdU-647 Cell Proliferation kit (Beyotime Biotechnology, Beijing, China) following the manufacturer's protocol.

The proliferation rates of rat peritoneal mesothelial cells were analyzed in this study by both the MTT and Edu staining methods. The MTT assay was carried out using the MTT (#M8180; Solarbio Life Sciences, Beijing, China) as instructed by the manufacturer. Briefly, rat peritoneal mesothelial cells were cultured in 96-well plates, mixed, and incubated with MTS reagent for 0.5–2 h at 37 °C in a humidified chamber with supply of 5% CO₂ and frequent shaking, followed by measuring the OD490 (absorbance at 490 nm) with a multi-functional plate reader. The evaluation of rat peritoneal mesothelial cell proliferation by Edu staining was finished using the BeyoClick EdU-647 Cell Proliferation kit (#C0081L; Beyotime Biotechnology, Beijing, China) following the manufacturer’s protocol.