Bioorthogonal photocatalytic proximity labeling for quantitative mapping of cell-cell interactions

Mapping dynamic cell-cell interactions with high sensitivity and spatial precision remains a central challenge in chemical biology. We present CAT-Cell, a photocatalytic proximity labeling strategy that utilizes visible-light-triggered unmasking of quinone methide (QM) probes to achieve selective covalent tagging of interacting cells under live-cell conditions. This protocol circumvents the requirement for genetic modification, allowing for the quantitative analysis of interactions between specific cell types, such as tumor and immune cells, without altering native cellular states. By integrating catalyst targeting with precise temporal light control, CAT-Cell ensures high spatiotemporal accuracy while minimizing perturbation to cellular physiology. We provide a comprehensive, step-by-step guide encompassing the chemical synthesis of probes and catalysts, cell surface functionalization, and quantitative flow cytometry analysis. CAT-Cell approach offers a general, sensitive, and tunable platform for mapping cellular interactomes in complex biological systems.