A SLC35C2 Transporter‐Targeting Fluorescent Probe for the Selective Detection of B Lymphocytes Identified by SLC‐CRISPRi and Unbiased Fluorescence Library Screening

T and B lymphocytes are two major adaptive immune cells in the human defense system. To real‐time monitor their diverse functions, a live‐cell‐selective probe for only one cell type is need to investigate the complex interaction of the immune cells. Herein, a small‐molecule probe CDyB for live B cells is developed by an unbiased fluorescence library screening. The cell selectivity was confirmed by multiparametric single‐cell analysis using CyTOF. Through a systematic SLC‐CRISPRi library screening, the molecular target of CDyB was identified as SLC35C2 transporter based on a gating‐oriented live‐cell distinction (GOLD) mechanism. The gene expression analysis and knock‐out experiments validated that the SLC35C2 transporter was the target for CDyB distinction. Interestingly, when CDyB was applied to study B cell development, the CDyB fluorescence and SLC35C2 expression were positively correlated with the B cell maturation process, and not involved in the T cell development. Through a systematic SLC‐CRISPRi library screening, the molecular target of CDyB was identified as the SLC35C2 transporter based on a gating‐oriented live‐cell distinction (GOLD) mechanism. Gene expression analysis and knock‐out experiments confirmed that the SLC35C2 transporter acted as the target responsible for the selectivity of CDyB for B‐cells over T‐cells.