Cells were plated in 96-well dishes and pre-treated with vehicle or R428
(1uM). A novel high-throughput irradiator utilizing a 50 kVp x-ray beam spectrum
was used to deliver 4 gray (Gy) as previously described (33 (link), 34 (link)). Cells were
fixed in 4% paraformaldehyde 4 hours later, permeabilized in 90% methanol,
blocked, and incubated with γ-H2AX primary antibody (1:500) overnight.
Cells were washed and incubated with FITC conjugated secondary antibody (1:1000)
(Santa Cruz Biotechnology). γ-H2AX fluorescence per cell was evaluated
via a SpectraMax i3 plate reader with MiniMax 300 imaging cytometer using
SoftMax Pro v6.4 software (Molecular Devices, Sunnyvale, CA, USA). All
γ-H2AX fluorescent values were averaged and then normalized to averaged
values from vehicle treated cells.
(1uM). A novel high-throughput irradiator utilizing a 50 kVp x-ray beam spectrum
was used to deliver 4 gray (Gy) as previously described (33 (link), 34 (link)). Cells were
fixed in 4% paraformaldehyde 4 hours later, permeabilized in 90% methanol,
blocked, and incubated with γ-H2AX primary antibody (1:500) overnight.
Cells were washed and incubated with FITC conjugated secondary antibody (1:1000)
(Santa Cruz Biotechnology). γ-H2AX fluorescence per cell was evaluated
via a SpectraMax i3 plate reader with MiniMax 300 imaging cytometer using
SoftMax Pro v6.4 software (Molecular Devices, Sunnyvale, CA, USA). All
γ-H2AX fluorescent values were averaged and then normalized to averaged
values from vehicle treated cells.
