Beyoclick edu kit

Cell proliferation was assessed using an EdU staining using the BeyoClick EdU kit (Beyotime Institute of Biotechnology), according to the manufacturer's protocol. Briefly, cells were treated with PBS, CDDP, MG132, or CDDP + MG132 for 48 h and were then incubated with 10 µM EdU at 37˚C for an additional 2 h. Following washing with PBS, cells were fixed with 4% polyformaldehyde at RT, permeabilized with 0.3 Triton X-100 followed by incubation with Click additive solution (Beyotime Institute of Biotechnology) at RT for 30 min in the dark. After staining with EdU, cells were counterstained with Hoechst 33342 for 10 min at RT and observed under a Leica fluorescence microscope DMi8 (Leica Biosystems; magnification, x200).

A BeyoClick™ EdU kit (Beyotime Institute of Biotechnology) was used to detect cell proliferation. Cells were incubated with 50 µM EdU solution for 2 h at 37°C. After fixing with 4% paraformaldehyde for 15 min at room temperature and permeabilizing with 0.2% Triton X-100 for 10 min, cells were stained with click reaction solution in the dark for 30 min and counterstained with Hoechst 33342 for 30 min at room temperature. EdU positive cells were counted using a fluorescence microscope (×20 magnification; Olympus Corporation).

The proliferative capability of cells was assessed by a BeyoClick™ EdU kit (Beyotime). In brief, cells were seeded in 24-well plates in which a cover glass had been placed in advance and cultured for 24 h. Then, cells were incubated with 10 μM EdU for 2 h at 37 °C and fixed in 4% formaldehyde for 15 min at RT. Triton X-100 (0.3%) was used to permeabilize the cells for 10 min. After that, the cells were incubated with Click Reaction Mixture (100 μL/well) for 30 min at RT, and DAPI was used to stain nuclei. The percentage of positive cells was calculated under a fluorescence microscope (Nikon, Japan) to reflect cell proliferation.

The BeyoClick EdU kit (Beyotime) was adopted as per the protocol provided by the manufacturer to detect DNA synthesis in cells. In short, 1×10⁵ cells after transfection and/or Mtb infection were incubated with EdU (10 μM) for 3 hours, and then fixed for 15 minutes with 4% paraformaldehyde. The cells were washed with phosphate-buffered saline (PBS) for 5 minutes and treated for 15 minutes with 0.3% Triton X-100. Then cells were incubated for 30 minutes with Click Additive solution in the dark. The nuclei were stained with 4ʹ,6-diamidino-2-phenylindole. The cells were observed under a fluorescence microscope. The proportion of EdU-positive cells was counted by ImageJ.