Phospho histone h3 serine 10 antibody

Cells were harvested from a single well of a 6-well plate using trypsin and washed 1x in PBS before fixation with dropwise addition of ice-cold 70% ethanol. After fixation on ice (or storage at −20°C) cells were washed 1x in PBS and permeabilized in 0.25% Triton X-100/PBS solution on ice for 15 minutes. Cells were washed 1x in PBS and resuspended in 1% bovine serum albumin/PBS solution containing 1µl/sample of Phospho-histone H3 (Serine 10) antibody conjugated to Alexa Fluor 488 (DC28, Cell Signaling). After washing 1x in PBS cells were resuspended in a solution of PBS containing 50µg/mL Propidium iodide (Santa Cruz Biotechnology) and 100µg/mL RNAse A (Roche). Flow cytometry was performed on a FACSCalibur (BD Biosciences) and analysed using FlowJo software. Single cells and G1/S/G2 peaks were manually gated. The gates for phospho-H3 positive cells were chosen by comparison to a population in which the antibody was omitted during processing.