Padronização de ensaios de apoptose e eferocitose in vitro no Laboratório de Imunofarmacologia - UFPB

Abstract

Multicellular organisms require the elimination of dysfunctional, senescent, infected, or damaged cells, and various forms of programmed cell death facilitate this process. Apoptosis represents one of these mechanisms, initiating through two converging pathways that activate effector caspases. Effector caspase-3 initiates the cleavage of several proteins that promote death by apoptosis. Apoptotic cells (ACs) are promptly cleared by phagocytes in a process called efferocytosis, which is crucial for maintaining tissue homeostasis. Deficiencies in efferocytosis have been associated with the development of inflammatory and autoimmune diseases. In this study, we standardized the generation of apoptotic cells through ultraviolet C (UVC) irradiation and assessed the efferocytosis by macrophages. HL-60 cells were exposed to UVC for five minutes and then incubated for two or four hours before evaluating cell viability and apoptosis. Reduced cell viability, increased phosphatidylserine exposure (detected by Annexin-V labeling), and caspase-3 activation were prominent at 4 hours post-irradiation, indicating an optimal time for generating apoptotic HL-60 cells for efferocytosis assays. When co-culturing apoptotic HL-60 cells (ACs) with Raw 264.7 cells (Mac), we noted that macrophages effectively efferocytosed the ACs, with an average efferocytosis of 61% in the 3 ACs:1 Mac condition, and 90% in the 5 ACs: 1Mac condition. Herein, we demonstrate a successful protocol for HL-60 apoptosis and efferocytosis by Raw 264.7 cells. Therefore, from this work it will be possible to develop projects about the in vitro efferocytosis process, allowing the immunopharmacology laboratory to evaluate the immunomodulatory potential of several compounds with promising action in mode of inflammation, efferocytosis and tissue repair.