4.7. Caspase 3/7 Assay and PARP Cleavage

Cells were treated according to the outlined protocol. Whole-cell lysates were prepared as described and incubated with Caspase 3/7 substrate (BD Pharmingen) for 60 min at 37 °C in a 96-well plate (OptiPlate, Perkin Elmer) and protein concentration was determined by a Bradford Assay (Roth). Substrate cleavage was measured for 50 cycles with 10 s delay (excitation at 380 nm, emission at 430–460 nm, Victor X4, Perkin Elmer). Kinetics were measured and calculated to the amount of protein per sample. Cells were treated according to the outlined protocol. For PARP cleavage analysis the samples were precipitated by acetone, boiled in 3× SDS-sample buffer for 10 min at 95 °C, and subjected to SDS-PAGE and Western blot analysis, using anti-Actin C4 (Sigma) and PARP polyclonal (Cell Signaling).