Beyoclick edu 555 cell proliferation assay kit

RC48 was purchased from RemeGen Co. Ltd. (Shandong, China). Dabrafenib was purchased from Selleck (Houston, USA). Annexin V-FITC/PI cell apoptosis assay kit, Cell Cycle Assay Kit, BeyoClick™ EdU-555 cell proliferation assay kit, TUNEL assay kit, and crystal violet staining solution were purchased from Beyotime Biotechnology Inc (Shanghai, China).

Cell proliferation was detected with the BeyoClick™ EdU-555 Cell Proliferation Assay Kit (C0075S, Beyotime). The cells were incubated in 6-well plates, and EdU working solution (20 μM) pre-warmed at 37 °C was added to the 6-well plates for a 1-h incubation. After the EdU labeling of cells was completed, the culture medium was removed. The cells were fixed by adding 1 mL of 4% paraformaldehyde for 15 min, incubated with phosphate-buffered saline (PBS) permeabilization solution for 10–15 min, and with 0.5 mL of Click reaction solution for 30 min in the dark (all at room temperature). Cell nuclei were stained with Hoechst 33,342 staining solution (C1025, Beyotime), and then cell proliferation was observed under a fluorescence microscope.
CD8⁺ T cell proliferation in the co-culture system was assayed with the CellTrace™ CFSE Cell Proliferation Kit (C34554, Thermo Fisher), and CFSE density was measured by flow cytometry.

The proliferation of porcine SCs was assayed by a BeyoClick™ EdU-555 cell proliferation assay kit (C0075S, Beyotime). Briefly, the original cell medium was used to dilute 1 μl EDU to prepare EDU working fluid, the remaining cell medium was discarded, and 1 ml of fresh medium was added again. Then, the newly prepared EDU working solution was added, and the cells were incubated for 2 h at 37°C in the dark. After that, the SCs were fixed with 4% paraformaldehyde for 15 min, and after washing three times, the SCs were permeabilized with 0.3% Triton X-100 for 15 min. Then, the SCs were washed twice and incubated with 0.5 ml click additive solution in the dark for 30 min. Finally, the SCs were counterstained with 1 × Hoechst 33342 (10 μM) in the dark for 10 min, washed three times and imaged by fluorescence microscopy (TS2-S-SM, Nikon). The images were analyzed by NIH ImageJ software (National Institutes of Health, Bethesda, MD, Unites States).

Cell proliferation was measured using the BeyoClick EdU-555 Cell Proliferation Assay Kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions. In short, on day 6, the embryos were transferred to fresh IVC medium containing 10% EdU with or without WDL and then incubated at 38.5 °C in an atmosphere of 5% CO₂ and 100% RH for 8 h. Next, the blastocysts were fixed with 3.7% paraformaldehyde in PBS-PVA for 30 min, and then permeabilized in 0.1% Triton X-100 at RT for 30 min. After washing four times with PBS-PVA, the embryos were incubated with 5% BeyoClick Additive Solution at 38.5 °C in an atmosphere of 5% CO₂ and 100% RH for 14 h. Then, the embryos were incubated with 10 mg/mL of Hoechst 33342 at RT for 10 min to label the nuclei. Finally, the embryos were washed four times with PBS-PVA and mounted on glass slides. An inverted fluorescence microscope (Ti2eU; Nikon, Tokyo, Japan) and ImageJ software (NIH) were used to count the number of EdU-positive cells and the total cells. The proliferation rate was calculated as the ratio of EdU-positive cells to the total number of cells.

The manufacturer’s instructions were followed to analyze cell proliferation using the BeyoClick EdU-555 Cell Proliferation Assay Kit (Beyotime, Shanghai, China). In short, on day 6 of the oocyte culture, 10% EdU was added to the IVC culture medium with or without CHE and then incubated in the dark at 38.5 °C in an atmosphere of 5% CO₂ for 10 h. After incubation, the blastocysts developed to day 7 were washed four times with PBS-PVA, fixed in PBS-PVA containing 3.7% paraformaldehyde for 30 min, and then containing 0.1% Triton X-100 permeabilized for 30 min, all at room temperature. After permeabilization, the blastocysts were washed four times, mixed with 5% BeyoClick Additive Solution, and incubated in the dark at 38.5 °C in an atmosphere of 5% CO₂ for 15 h. Finally, the blastocysts were incubated with 10 µg/mL Hoechst 33,342 in the dark at 37.5 °C for seven minutes to mark the nuclei. An inverted fluorescence microscope (Ti2eU; Nikon, Tokyo, Japan) and ImageJ version 8.0.2 software (NIH, Bethesda, MD, USA) were used to count the number of EdU-positive cells and total cells. We compared the level of cell proliferation by calculating the percentage of EdU-positive cells in the total number of blastocysts. The number of blastocysts on day 7 for the EDU staining assay was 15 to 20, and the assay was repeated more than three times.