Edu medium

A total of 1 × 10⁶ microglia were harvested and placed into the upper chamber of a 12-well trans-well plate (0.65 µm, Corning, New York, NY, USA), containing 1 µg/ml LPS. In addition, 1 × 10⁶ NSCs were placed into the lower chamber, wells contained either culture medium; 40 nM leukosomes; or 40 nM lnc-EPS-leukosomes (n = 8). The microglia and medium in the control group were not untreated.
After culturing for 24 h at 37 °C, NSCs were cultured overnight using EdU medium (Ribobio, Guangzhou, China) and treated with 0.3% Triton X-100 in 1% fetal bovine serum (FBS) for 15 min at 37 °C. Then, cells were washed with PBS and to stain mitotic NSCs, 100 μL 1 × Apollo-567 (Ribobio, Guangzhou, China) was added to each well. DAPI was used to stain nuclei. Cells were evaluated and imaged using a fluorescence microscope (DMI4000B, Leica, Germany) at 565 nm and 461 nm.
The grouping and stimulation methods were identical as mentioned above. After 48 h, NSC were harvested, washed, and resuspended in PBS. A FITC-conjugated Annexin V Apoptosis Detection Kit (BD Biosciences, Franklin, NJ, USA) was used to identify apoptotic NSCs. Procedures were strictly performed by following the kit instructions, and cell apoptosis was evaluated by FACScan laser flow cytometer (CyFlow^® Cube, Partec, Germany).

EdU staining was performed using a Cell-Light EdU Apollo 488 In Vitro Kit (RiboBio, Guangzhou, China). For the EdU assay, ICP-1 cells were treated with EdU medium (1:1,000; RiboBio, Guangzhou, China) for 2 h at 37°C and then fixed in 4% paraformaldehyde solution (Yike, Guangzhou, China) for 30 min. The cells were subsequently permeabilized using 0.1% Triton X-100 solution (Gbico, New York, United States). The cells were incubated with the staining solution for 30 min at room temperature in the dark. Finally, the stained cells were scraped off with a cell scraper, collected in a 1.5 mL centrifuge tube and resuspended in 1 mL PBS. A BD Accouri C6 flow cytometer (BD Biosciences, San Jose, CA, United States) was utilized for the analysis of stained cells (16 (link)).

Cells were seeded to 96-well plates which were added with the EdU medium (Ribobio, Guangzhou, China) in PBS, then fixed with 4% paraformaldehyde for fixing. Cells were cultured with DAPI solution for 30 min and observed under a fluorescence microscope (Olympus, Tokyo, Japan).

A549 and H226 cells were inoculated into 24-well plates, respectively, and cultured for 24 h. After that, the cells were incubated with 200 μL of 50 μmol/L EdU medium (RiboBio Co., LTD, Guangzhou, China) for 2 h, rinsed with PBS and then fixed with paraformaldehyde for 10 min. Following that, the cells were incubated for 5 min with 200 μL of 2 mg/ml of glycine and rinsed with PBS for 5 min. One hundred microliters of PBS with 0.5% Triton X-100 was added to each well, and the cells were placed on a shaker for 10 min and then washed with PBS for 5 min. Subsequently, the cells were stained with Apollo® solution in the dark for 30 min, and nuclear DNA was counterstained with DAPI. Then, the cells were observed, and the images were collected using a fluorescent microscope in the dark.

5×10³ BC cells seeded and grown in 96-well plates overnight and 100 μL 50 μM EdU medium (RiboBio, Guangzhou, China) per well were cultured for 2 h. Then the cells were fixed by methanol for 30 min and washing with PBS for 5 min twice. After permeabilizing with 0.5% TritonX-100 for 10 min twice and washing with PBS for 5 min, 1×Apollo dye was used to stain the cells for 30 min, repeated washing. Finally, the signal was visualized and recorded by a microscope after Hoechst 33342 counterstaining.