Development of a Mature B Lymphocyte Probe through Gating-Oriented Live-Cell Distinction (GOLD) and Selective Imaging of Topical Spleen.

B lymphocytes play a pivotal role in the adaptive immune system by facilitating antibody production. Young B cell progenitors originate in the bone marrow and migrate to the spleen for antigen-dependent maturation, leading to the development of diverse B cell subtypes. Thus, tracking B cell trajectories through cell type distinction is essential for an appropriate checkpoint assessment. Despite its significance, monitoring specific B cell subclasses in live states has been hindered by a lack of suitable molecular tools. In this study, we introduce CDoB as the first mature B cell-selective probe, enabling real-time discrimination of three classified stages in B-cell development: progenitor, transitional, and mature B cells, through a single analysis using CyTOF. The selective mechanism of CDoB , elucidated as gating-oriented live-cell distinction (GOLD), targets SLC25A16, identified through systematic screening of SLC-CRISPRa and CRISPRi libraries. CDoB selectively brightens mature B cells in the mitochondrial area using SLC25A16 as the main gate, and the staining intensity correlates positively with the expression level of SLC25A16 along the B cell maturation continuum. In spleen tissues, CDoB demonstrates selective marking in mature B cell areas in live tissue status, representing the first performance achieved by a small-molecule fluorescent probe.