Anti phospho histone h3 ser10

Cells were synchronized as described above in a 6 well plate. Interphase cells were trypsinized, washed once in PBS, and pelleted. Mitotic cells were isolated by shake off, spun down, and washed once in PBS. To fix cells, the cell pellet was resuspended in 500 μL PBS then 9.5 mL −20°C ethanol was added to the cell drop wise while lightly vortexing, and incubated on ice for >30 minutes. Following fixation, cells were washed once in PBS+0.3% BSA (w/v), then once in PBS + 3% BSA (w/v) + 0.1% Triton X-100 (v/v), and then blocked using antibody dilution buffer (AbDil, 20 mM Tris-HCl, 150 mM NaCl, 0.1% Triton X-100, 3% bovine serum albumin, 0.1% NaN₃, pH 7.5) on ice for 30 minutes. After blocking, cells were resuspended in 1:1000 anti-phospho-histone H3 Ser10 (Abcam, ab5176) diluted in AbDil and incubated overnight at 4°C, rotating end over end. Cells were then washed once with PBS + 3% BSA + 0.1% Triton X-100, and incubated in 1:300 goat anti-rabbit cy5 (Jackson ImmunoResearch Laboratories) diluted in AbDil for 1 hour on ice. Cells were then washed once with PBS + 3% BSA + 0.1% Triton X-100, and incubated with PBS + 20 μg/mL hoescht for >30 minutes on ice. Cells were then strained and measurements were made using the BD LSRFortessa Cell Analyzer (BD Biosciences). Data was analyzed using FlowJo. We gated for live cells and singlets prior to any quantification.