Annexin V and caspase-3 apoptosis assays

Apoptotic cells were measured by using the Alexa Fluor 488 Annexin V Dead Cell Apoptosis kit (Invitrogen, Waltham, MA) according to the manufacturer’s protocol. Briefly, cells were plated (1 × 10⁵ cells per plate) on 10-cm plates and treated with the indicated agents for 6 days and allowed to recover for 4 days. Cells were collected at the end of treatment (6 days) or after treatment+recovery, washed with 1 × Annexin binding buffer and stained with a solution of Alexa Fluor 488 Annexin V and propidium iodide (100 μg ml⁻¹) as directed by the protocol. Samples were then analysed on the Beckman Coulter Gallios Flow Cytometer, and data were analysed with Kaluza software to obtained the percentages of Annexin V-positive/propidium iodide-negative (early apoptosis) and Annexin V-positive/propidium iodide-positive (late apoptosis) cells.

For the caspase-3 activity assay, following drug treatment, cells were collected, washed with a solution of PBS with 2% FBS and fixed with 100 μl of the fixation solution from the Cytofix/CytoPerm kit (BD Biosciences). Cells were then washed with Perm/Wash buffer and stained with phycoerythrin-conjugated rabbit active caspase-3 antibody (BD Biosciences) for 30 min on ice. Cells were finally washed, resuspended in the PBS with 2% FBS solution and analysed on the Becton Dickinson FACS Calibur Flow Cytometer to obtain the percentage of caspase-3-positive cells.