Anti p histone h3 antibody c 2

Cells were disassociated using 0.25% Trypsin/EDTA (nacalai tesque, 35554-64), washed with 1% BSA/PBS, and fixed in 1% PFA on ice for 1 h. After washing with 1% BSA/PBS, the cells were permeabilized/blocked with 1% Triton-X-, 1% BSA- and 10% FBS-contained PBS for 1.5 h at room temperature. They were then incubated overnight at 4°C with appropriate amounts of primary antibodies, including the Anti-p-Histone H3 antibody (C-2) (1:100, Santa Cruz, sc-374669) and Mouse mAb IgG_2b Isotype Control monoclonal antibody (1:100, Santa Cruz, sc-3879) in 1% FBS-, 1% BSA- and 0.1% Triton-X-contained PBS. On the following day, after washing with 0.1% Triton/PBS, the cells were incubated with fluorescent-labeled IgG (H + L) secondary antibodies (Abcam, Goat anti-mouse, Alexa Fluor 594) at 1:500 dilution in 1% FBS-, 1% BSA- and 0.1% Triton-X-contained PBS for 2 h at room temperature. Immediately after washing with 0.1% Triton/PBS, the cells were resuspended in an ice-cold Sheath fluid (Sysmex, CG-974-836) at 1 × 10⁶ cells/mL and analyzed on a flow cytometer. FACS analysis was performed using Sysmex RF-500 with FlowJo (BD Biosciences), and a minimum of 10,000 events were recorded for each sample. Fluorescence minus control was used to gate the subpopulations.

For immunofluorescence studies, cells grown on a 4-well plate were fixed with freshly prepared 4% paraformaldehyde in PBS for 15 min at room temperature and permeabilized and blocked with 1% Triton-X, 1% BSA, and 10% FBS-contained PBS for 1 h at room temperature. The cells were incubated with primary antibodies in 10% FBS, 1% BSA, and 1% Triton-X-containing PBS overnight at 4°C. On the following day, the cells were washed three times in 0.1% Triton/PBS and incubated with fluorescent-labeled IgG (H + L) secondary antibodies (Abcam, Goat anti-mouse (Alexa Fluor 488): ab150113, (Alexa Fluor 594): ab150116, Goat anti-rabbit (Alexa Fluor 594: ab150080) at 1:500 dilutions in 1% FBS, 1% BSA, and 1% Triton-X-containing PBS for 2 h at room temperature. The cells were washed three times and filled with 0.1% Triton-containing PBS, and nuclei were counter-stained with DAPI (Sigma-Aldrich, D9542) for immunofluorescence. Primary antibodies used in this study include Anti-Myosin Heavy Chain antibody (MF20) (1:500, R&D Systems, MAB4470), Anti-Myogenin antibody (1:500, Abcam, ab124800), Anti-p-Histone H3 antibody (C-2) (1:1000, Santa Cruz, sc-374669), and Anti-Cleaved Caspase-3 antibody (Asp175) (5A1E) (1:500, Cell Signaling Technology, #9664). The observation was performed under a microscope (Keyence, BZ-X710).