For BrdU and pS10-H3 fluorescence-activated cell sorting (FACS) analysis, ES cells were singularized with Accutase®. Cells were washed with cold PBS and fixed in 70% ethanol at 4°C overnight. Cells were pelleted and treated with 2 N HCl/Triton X-100 at room temperature for 30 min to denature DNA. After neutralization, cells were incubated with primary antibody for 30 min in FACS buffer and washed in PBS. Antibodies APC anti-BrdU (BioLegend, #339808, 1:500 dilution) and anti-pS10-H3-Alexa488 (Cell Signaling Technology, #3465S, 1:1000 dilution) were used. After 3 washes in cold PBS, cells were resuspended in PBS and analyzed by BD FACSAria™ Fusion.
For cell-counting experiments, cells were treated with Accutase® and counted and seeded at the same number before counting. The following day, cells were rinsed with PBS, singularized with Accutase®, and harvested. After pelleting, cells were resuspended in PBS. Exactly 50 μL of Sphero™ ACCUCOUNT fluorescent particles (Spherotech Inc., #ACFP-70-10) were added into each sample. Mixed samples were analyzed by BD FACSAria Fusion.
For cell-counting experiments, cells were treated with Accutase® and counted and seeded at the same number before counting. The following day, cells were rinsed with PBS, singularized with Accutase®, and harvested. After pelleting, cells were resuspended in PBS. Exactly 50 μL of Sphero™ ACCUCOUNT fluorescent particles (Spherotech Inc., #ACFP-70-10) were added into each sample. Mixed samples were analyzed by BD FACSAria Fusion.