Brdu labeling solution

For BrdU binding, remove the existing culture medium from the cells and replace with 10 µM BrdU labeling solution (Abcam, U.S.A.). Incubate the cells in the BrdU labeling solution at 37°C for 12 h and then immunofluorescence staining was used. Fix cells using 4% paraformaldehyde in 0.M PBS for 15 min at room temperature. For sample permeabilization, using 0.3% Triton X-100 for 15 min. For DNA hydrolysis, incubate cells in 1 M HCL for 10 min at 37°C. Then incubate cells with 3% BSA for 20 min to block the possible unspecific binding of the antibodies. Incubate cells with diluted BrdU-antibody (Abcam, U.S.A.) in 1% BSA overnight at 4°C. Then incubate cells with secondary antibody Cy3-goat anti-mouse (Abcam, U.S.A. and mounting the slides with Mountain medium with DAPI (Blue). Images were collected from at least three independent cultures per group were used for statistical analysis [11 (link)]. For CCK-8 assay, cells were seeded to 96-well plates with 2000 cells/well and incubated for 1, 2, 3, 4, and 5 d. Total 10 µl of CCK-8 solution was added to each well of the 96-well plate. Then incubate the plate for 2 h in the incubator. Measure the O.D. at 450 nm using a microplate reader.

We performed a BrdU incorporation assay using the method described in the instruction manual of the BrdU staining and BrdU assay protocol from Abcam (Cambridge, UK). Astrocytes were plated at a density of 1 × 10⁵ cells/mL on Poly-L-lysine-coated coverslips in 24-well plates and then incubeted with serum-starved DMEM for 6 h. The cells were treated with or without S-equol (1–100 nM) together with 10 µM BrdU labeling solution (Abcam) for 24 h. The cells were then washed with PBS and fixed with 4% PFA. The DNA was hydrolyzed using 2 M HCl for 1 h at room temperature and then blocked with 2% FBS. The cells were incubated with rat anti-BrdU antibody [BU1/75 (ICR1)] (ab6326) (1:200; Abcam) and a donkey anti-rat IgG (H + L) secondary antibody, Alexa Fluor^® 594 conjugate (1:200; Thermo Fisher Scientific, Inc.). Cell nuclei were also stained with DAPI. The cells were then inspected under a laser confocal scanning microscope (Zeiss LSM 880, Carl Zeiss Microscopy GmbH).