Apollo 567 reaction cocktail

For the EdU assay, the C6 cells from the LV-NC and LV-BLBP groups were seeded into 24-well plates and 25-ml culture flasks at densities of 2×10⁴ or 2×10⁵ cells/well, respectively, in 10 or 1% FBS culture medium. After 24 h, the cells were treated with the EdU (50 µM) reagent (Ribobio, Guangzhou, China) for 1 h and stained with the Apollo-567 reaction cocktail (Ribobio) for 30 min. After washing thrice with PBS, the cells in the 24-well plates were counterstained with Hoechst for nuclear staining and observed under a microscope, while the cells in the 25 ml culture flasks were assessed using FACSCalibur instrument (BD Biosciences) to determine the proportion of EdU-positive cells, all results were from three independent replicates.

Cells were seeded at a density of 2×10⁵ cells/well in 6-well plates and incubated for 24 h. The cells were then treated with 50 µM EdU reagent (Guangzhou RiboBio Co., Ltd., Guangzhou, China) followed by Apollo-567 reaction cocktail (Guangzhou RiboBio Co., Ltd.) for 30 min. Following three washes with phosphate-buffered saline, cells were counterstained with Hoechst (dilution 1:1,000; Guangzhou RiboBio Co., Ltd.) for 10 min at room temperature for nuclear staining. All cells were observed using an EVOS FL Imaging System (Thermo Fisher Scientific, Inc.).