Alexa fluor 488 conjugated phosphorylated histone h3 antibody

Cells were drugged at 50–70% confluency for 48 h and fixed with neutral-buffered formalin for 10 min, followed by permeabilization with 0.125% Triton X-100 for 5 min. Cells were blocked with 10% bovine serum albumin (BSA) overnight. Cells were probed with a primary antibody against phosphorylated aurora kinase A/B/C (Antibody #2914; Cell Signaling Technology) at a 1:100 dilution followed by an Alexa Fluor-555 (Invitrogen) conjugated secondary antibody at a 1:400 dilution in blocking buffer. Cells were washed in PBS and further incubated with Alexa Fluor-488 conjugated phosphorylated histone H3 antibody (antibody #9713 1:100 dilution; Cell Signaling Technology). Cells were counterstained with Hoechst 33258 and mounted on slides. Confocal images were taken with the Zeiss LSM 510 meta-confocal microscope (Carl Zeiss, Thornwood, NY) using a 63× objective.

Cells were plated in 100 mm dishes and drugged at 50–70% confluency. Both floating and attached cells were collected 48 h after treatment, washed in PBS and fixed with 4% freshly made paraformaldehyde. Cells were then permeabilized with 90% cold methanol. Permeabilized cells were stained with Alexa Fluor-488 conjugated phosphorylated histone H3 antibody (1: 100 dilution; Cell Signaling Technology). Nuclei were stained with propidium iodide (Sigma) in PBS containing 1% BSA. Flow cytometry was performed on a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA). Cell cycle analysis was performed using BD FACSDiva software and FlowJo.