For staining of active caspase-3, cells were washed with PBS, fixed in 2% formaldehyde and permeabilised with 0.5% saponin (Sigma-Aldrich). Cells were incubated with anti-active caspase-3 (BD Pharmingen, Heidelberg, Germany) in PBS/0.5% BSA/0.5% saponin for 30 min, stained with anti-rabbit-Alexa-Fluor647 (Dianova GmbH, Hamburg, Germany) for 30 min and analyzed by flow cytometry.
In most experiments, cell death was assessed by Propidium iodide staining for loss of cell membrane integrity. Cells were harvested in culture media, and PI was added at a final concentration of 1 μg/ml before analysis by flow cytometry on a FACS Calibur. In some experiments, cell death by loss of cell membrane integrity was quantified using the LIVE/DEAD Fixable Far Red Dead Cell Stain (Molecular Probes, Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions.
Annexin V-Propidium iodide staining was done by washing cells with Annexin V-binding buffer (eBioscience) and staining with Annexin V-FITC (1 : 50; BD Pharmingen) 20 min at 4 °C. Propidium iodide (1 μg/ml; Sigma-Aldrich) was added and cells were analyzed on a FACS Calibur (Becton Dickinson, Heidelberg, Germany).
In most experiments, cell death was assessed by Propidium iodide staining for loss of cell membrane integrity. Cells were harvested in culture media, and PI was added at a final concentration of 1 μg/ml before analysis by flow cytometry on a FACS Calibur. In some experiments, cell death by loss of cell membrane integrity was quantified using the LIVE/DEAD Fixable Far Red Dead Cell Stain (Molecular Probes, Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions.
Annexin V-Propidium iodide staining was done by washing cells with Annexin V-binding buffer (eBioscience) and staining with Annexin V-FITC (1 : 50; BD Pharmingen) 20 min at 4 °C. Propidium iodide (1 μg/ml; Sigma-Aldrich) was added and cells were analyzed on a FACS Calibur (Becton Dickinson, Heidelberg, Germany).