Edu assay kit

Cell proliferation was examined using an EdU assay kit purchased from Beyotime Biotechnology (China) and a cell counting kit-8 (CCK8) assay from MedChem Express (USA). Briefly, the three stable cell lines (LV-NC, OE-LV-miR-3571, and OE-LV-CLDN1) were trypsinized then seeded into the wells of 96-well micro titerplates in DMEM supplemented with 10% FBS at an initial concentration of 1 × 10⁴ cells per well and incubated at 37°C in an atmosphere containing 5% CO₂ for 24 h. The medium was exchanged for DMEM supplemented with 1% FBS and the cells incubated for a further 24 h. The viability of the three stable cell lines was then examined in accordance with the manufacturer's instructions. The result of CCK8 assay were calculated from relative light absorbance while cells from the EdU assay were imaged using a fluorescence microscope (IX71 Olympus Corporation, Japan) and analyzed with image J software. Cell viability in EdU assay = numbers of cells with fluoresing red (stained with Azide 555)/ numbers of cells fluoresing blue (stained with Hoechst 33342).

The EdU assay kit was purchased from Beyotime Company (C0078S, China). According to the manufacturer’s protocol, transfected cells were treated with EdU reagent for 3h. Then, cells were fixed with 4% paraformaldehyde for 15 min at room temperature, followed by incubation with 0.3% Triton X-100 for 15 min at room temperature. Finally, cells were stained with fluorescent dye and Hoechst. The Nikon TI2-D-PD inverted microscope (Japan) was used to capture the images.

CCK-8 and colony formation assays were used to evaluate cell proliferation. For the CCK-8 assay, cells were seeded and cultured in 96-well plates for 48 h. The culture medium was replaced with CCK-8 solution, and the absorbance was measured at 450 nm after 4 h of incubation at 37 °C [22 (link)]. For the colony formation assay, cells were incubated in a 60-mm dish for two weeks and then fixed with paraformaldehyde for 20 min. After being washed with PBS, the colonies were stained with 0.5% crystal violet for 15 min. The cell proliferation was further evaluated by an EdU staining assay using an EdU assay kit (Beyotime) according to the following protocol. Briefly, in 12-well plates, 2 × 10^5 cells were seeded and incubated for 24 h. The cells were treated with 2% glycine and 0.5% Triton X-100 and stained with Apollo staining reaction buffer and DAPI. EdU-positive cells were analyzed by fluorescence microscopy.
Cell apoptosis was determined by flow cytometry assay as reported in previous studies [23 (link)]. In brief, cells were dissociated and resuspended at 1 × 10⁶ cells/mL in assay buffer and then cultured with 10 μL/ml JC-1 for 15 min at 37 °C. Cells were then analyzed by flow cytometry after washing twice with assay buffer.

The EDU assay was performed as described previously [22 (link)]. In short, cells (1 × 10⁵) were seeded in 24-well plates and cultured for 20 h. After incubation with EDU reagent for 2 h, cells were fixed with 4% paraformaldehyde (solarbio) and permeabilized with 0.5% Triton X-100 (solarbio). Sequentially, cells were counterstained following the manufacturer’s instructions of the EDU assay kit (Beyotime, Biotechnology, China). The images were captured using a laser scanning confocal microscope (Olympus) and then the EDU-positive cells percentage could be calculated.

Induced macrophages were cocultured with glioblastoma cells (LN229 and U251) in a Transwell chamber for 48 h. Induced macrophages were placed in the upper chamber and glioblastoma cells were placed in the lower chamber (Schematic diagram was shown in Supplementary Fig. 1D). After coculture, glioblastoma cells were obtained from the bottom chamber. An EdU assay kit (Beyotime, China) was used to determine the glioblastoma cells proliferation ability according to the manufacturer’s protocol.