Tritonx 100

Cell proliferation assays were performed using the Cell-Light^TM EdU Apollo In Vitro Kit (RiboBio, Guangzhou, China). Cancer cells (1 × 10⁵) were seeded in 96-well plates and treated according to the experimental requirements. Subsequently, EdU medium was added and the cells were incubated for 2 h. After washing the cells twice with PBS, they were fixed with 4% PFA solution (Biosharp) for 30 min. Cells were then incubated with glycine (2 mg/ml; Biofroxx, Germany) for 5 min and permeabilized with 0.5% Triton X-100 (Beyotime) for 10 min. After adding 1 × Apollo^® staining solution (RiboBio) and incubating for 30 min at light avoidance and RT, the cells were washed three times with 0.5% TritonX-100. Finally, a 1 × Hoechst 33342 reaction solution (RiboBio) was added and incubated for 30 min with light avoidance at RT. The cells were imaged using a microscope (Leica Microsystems) and counted in four randomly selected fields.

EdU assay was used to examine cell proliferation. Glioma cell lines in the logarithmic growth phase were seeded into 96-well plates at a density of 2×10³- 4×10⁴. After 24 h, the adherent cells to the wells were transfected. Five parallel wells were set up for each group. Cells in each well after transfection for 48 h were cultured with 100 μL EdU medium (RIBOBIO, China) for 2 h and fixed with 100 μL of cell fixation solution (PBS containing 4% polyformaldehyde) for 30 min at room temperature. Subsequently, the cells were incubated with 2 mg/mL glycine (Solarbio, China) for 5 min, rinsed with 100 μL of PBS containing 0.5% TritonX-100 (RIBOBIO, China) for 10 min, and stained using 1 × Apollo staining reaction solution (RIBOBIO, China) for 30 min in conditions devoid of light. Next, the cells were reacted with 100 μL of the 1 × Hoechst 33342 reaction solution (RIBOBIO, China) for 30 min and sealed with 100 μL of the anti-fluorescence quenching agent. Six to ten fields of view were randomly selected for each well and photographed under a fluorescence microscope.