Atomic force microscopy imaging of RBL-2H3 cell degranulation.

Understanding the biophysical mechanisms of degranulation is crucial for managing allergic diseases, yet the spatiotemporal coordination of membrane and cytoskeletal dynamics during these reactions remains incompletely understood. In this study, we utilized Atomic Force Microscopy (AFM) to conduct a time-resolved investigation of RBL-2H3 cells during anti-DNP IgE-induced activation. By systematically mapping the cell surface, we quantified phase-specific changes in morphology, surface adhesion, and cortical stiffness (Young's modulus). Our results reveal a striking temporal asynchronicity, with cell height and surface adhesion peaking at 8 h, reflecting receptor-driven membrane ruffling and sensitization. In contrast, the Young's modulus reached its maximum at 12 h, indicating a delayed mechanical reinforcement driven by profound cytoskeletal rearrangement for active granule transport. Furthermore, we characterized the collapsed-sphere ultrastructure of secreted extracellular vesicles (EVs). These findings successfully decouple the initial membrane sensitization from the subsequent intracellular execution phase, identifying novel nanomechanical biomarkers to understand vesicle-mediated communication and to guide the design of stage-specific therapeutic interventions.