Mouse anti active caspase 3

The apoptotic response after genotoxic drug treatment was measured using flow cytometry for sub-G1 determination. Supernatant and attached cells were collected, washed once with PBS and fixed in 70% ethanol. Ethanol-fixed cells were stained with propidium iodide (PI) at room temperature for 1 h in PBS containing 20 μg/ml PI (Sigma–Aldrich), 200 μg/ml RNase A, and 0.1% Triton X-100. The percentage of sub-G1 cells was calculated using the CytoSoft software (Millipore, Billerica, MA, USA). For γH₂AX and active caspase-3 immunostaining, cells were fixed with 1% formaldehyde and then with 70% ethanol. Afterwards, the cells were blocked, permeabilized, incubated with either primary mouse monoclonal antibody to γH2AX (Ser-139) (Upstate Biotechnology, Lake Placid, NY, USA) and diluted 1:500, or mouse anti-active caspase 3 (BD, Pharmigen, San Diego, CA, USA) diluted 1:50 for 2 h at room temperature. This was followed by incubation with anti-mouse FITC secondary antibody (Sigma-Aldrich) that was diluted 1:200 for 1 h at room temperature. The percentage of γH₂AX positive cells was again calculated using the CytoSoft software (Millipore).

1.5 × 10⁵ cells were plated in a 12 multi-well plate and incubated with 3 μM cisplatin for 6, 24 and 72 h. Supernatant and attached cells were collected, fixed with 1% formaldehyde and then with 70% ethanol. Afterwards, cells were blocked, permeabilized and incubated with a primary mouse monoclonal antibody to γH2AX (Ser-139) (Upstate Biotechnology, Lake Placid, NY, USA) diluted 1:500 for 16 h at 4 °C, followed by incubation with anti-mouse FITC secondary antibody (Sigma-Aldrich) diluted 1:200 for 2 h at room temperature, or with a mouse anti-active caspase 3 (BD, Pharmigen, San Diego, CA, USA) diluted 1:50 for 16 h at 4 °C. In both cases, cells were then stained with propidium iodide (PI) at room temperature for 1 h, in PBS containing 20 μg/mL PI (Sigma-Aldrich), 200 μg/mL RNase A and 0.1% Triton X-100.