Evaluation of an intracellular magneto-mechanical approach for regulating dendritic cell activation.

Dendritic cells (DCs) are crucial in processing and cross-presenting tumor-associated antigens via MHC I molecules to prime CD8+ T lymphocytes (CTLs) for antitumor immunity. However, the moderate regulation of antigen endocytic escape from lysosomes into the cytoplasm hinders this cross-presentation process due to the lack of finely tunable tools at the organelle-specific level. Magneto-mechanical modulation technology provides a promising approach to regulate lysosomal membrane permeability (LMP) and promote antigen leakage for efficient cross-presentation. This approach relies on magnetic nanorobots within lysosomes that can exert adjustable intracellular forces in response to external rotating magnetic fields. This chapter presents a comprehensive protocol for evaluating the antitumor response of DCs induced by magneto-mechanical regulation. This protocol includes a step-by-step assessment of cytotoxicity on DCs caused by magneto-mechanical regulation, analysis of nanorobot colocalization with lysosomes, and their responses (such as LMP, cytoskeleton morphology, and mitochondria function) through fluorescence imaging. Additionally, we quantify DC mechano-activation and cross-presentation efficiency using flow cytometry.