Annexin 5 apc detection kit

Apoptosis was detected with an Annexin-V APC detection kit (eBioscience, USA). Briefly, cells subjected to different treatments were harvested and incubated with anti-Annexin V antibody labeled with APC and PI for 10 min in the dark, following the protocol provided by the manufacturer. Apoptotic cells were quantified using FACS Calibur flow cytometry.

Caspase 3/7 activity of the cancer cells after lentivirus infection was assessed with Caspase-Glo® 3/7 Assay Systems (G8091, Promega, USA) following the instructions of product manual.
Early apoptosis events were detected with Annexin-V APC detection kit (Cat. 88-8007, eBioscience, USA) following the instructions of product manual. Fluorescence-activated cell sorting was performed using Guava easyCyte HT cytometry system (Millipore, USA). Statistical significance was tested using t-test.

Cells (100,000) were incubated with the transfection agent and PPRH (HpsPr-C) for 48 h. Then, cells were trypsinized, collected in PBS, and centrifuged at 1200× g for 5 min. The resulting pellet was resuspended in 100 μL of Binding Buffer 1× and incubated with 5 μL of the APC Annexin V Detection Kit (Invitrogen, Thermo Scientific, Barcelona, Spain) for 15 min at room temperature. After a 5 min centrifugation at 1200× g, the pellet was washed with 1 mL of Binding Buffer 1×. The samples were centrifuged for 5 min, resuspended in a final volume of 500 μL of Binding Buffer 1×, and propidium iodide was added to achieve a concentration of 5 μg/mL (Merck, Madrid, Spain). Apoptosis analyses were performed using a Gallios flow cytometer (Beckman Coulter, Inc., Barcelona, Spain) from the CCiTUB.