Annexin 5 fitc apoptosis staining

Analysis was done following the specifications of the kit Annexin V-FITC apoptosis Staining (Abcam, Cambridge, UK, #ab14085) and the optimal concentrations were optimized before the experiments. Hep3B cells were seeded in a 6-well plate, incubated overnight and starved (≥4 h at 0% FBS) before treated with TGF-β for 48, 72 h. Some conditions were incubated with Q-VD-OPH 1 h before the treatment with TGF-β to inhibit the caspases. At the precise time, attached cells, after trypsinization, and those in culture medium were collected and added to a flow cytometry tube containing 1 mL of Annexin Binding Buffer (ABB). Tubes were centrifuged at 480g for 5 min and the supernatant discarded. Then, cells were resuspended in 100 μL of ABB with 6 μL of Annexin V-APC and 5 μL of Propidium Iodide (PI) and incubated for 15 min at room temperature in the dark. Finally, 100 μL of ABB are added, and the reading is done. Flow cytometry was performed by using a BD FACS Canto II Cytometer and analyzed by BD FACSDiva ™ v9.0 Software (BD Biosciences, San Jose, CA, USA). BD FlowJo™ v10.8.1 Software (BD Biosciences, San Jose, CA, USA) was used for the generation of graphs. All was performed at the Scientific and Technological Centers of the University of Barcelona (CCiTUB).