Arrayscan vti hcs instrument

HeLa FlpIn T-REx cells were
arrested in early S-phase in growth media containing 2 mM thymidine
(Sigma) for 16 h. Cells were then released for 8 h in thymidine-free
media, and these were then arrested again with thymidine for 16 h
followed by the release in thymidine-free media. Cells were treated
for 12 h with DMSO or compounds, and then these were fixed in 3.7%
formaldehyde (Agar Scientific). Cells were permeabilized with 0.1%
Triton X-100, and then these were incubated with anti-phospho-histone
H3 (Ser10) antibody (Abcam ab5176). The cells were washed with PBS,
and then these were incubated with Alexa Fluor 488-labeled goat anti-rabbit
IgG (Invitrogen A11034) in the presence of 4 μg/mL Hoechst 33342
(Invitrogen H3570). Cells were washed in PBS, and then these were
imaged on an Arrayscan VTi HCS instrument (Thermo Fisher) using the
Target Activation V4 BioApplication. Mitotic cells were scored as
phospho-histone H3-stained cells per 2000 Hoechst 33342-stained nuclei,
as described in Ibbeson et al.^{27 (link)}