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

Abstract

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.

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

“…The CDoB ++ populations included mature B cells (FO B, MZ B, recirculating B), whereas CDoB + contained transitional B cells, and CDoB– consisted of B cell progenitors (pro-B, pre-B, and immature B), T, and NK cells (Figure D). This performance contrasts with previously reported B cell probes, CDgB and CDyB , which have low sensitivity to monitor overall B cell stages from BM to SP. This result underscores the efficacy of CDoB as an ideal tool for tracking B cell maturation, especially in selectively highlighting mature B lymphocytes, leading to its designation as C ompound D esignation for o ld B .…”

“…With these fluorescent probes, we can not only monitor dynamic cellular states but also uncover intrinsic cellular activities without disturbance. 1 We have developed two B cell selective probes CDgB 2 and CDyB, 3 which exhibit specificity over T cells. While we achieved general selectivity for B lymphocytes, we recognized the need to differentiate specific B cell subsets.…”

“…We have developed two B cell selective probes CDgB ( 2 ) and CDyB , 3 which exhibit specificity over T cells. While we achieved general selectivity for B lymphocytes, we recognized the need to differentiate specific B cell subsets.…”

Synthesis and Application of Two-Photon Active Fluorescent Rhodol Dyes for Antibody Conjugation and In Vitro Cell Imaging

“…First-generation rhodols ( 2 ) were synthetized containing a diethylamino group on one side of the xanthene core (Figure A). However, there was still some room for improvement in fluorescence properties, and therefore, on the one hand, an electron-donating julolidine core − was incorporated into next-generation compounds ( 3 ) that inhibits the internal rotation of the amino group, decreasing the twisted intramolecular charge transfer (TICT). On the other hand, in some cases, the π-system was extended ( 4 ) to fine-tune the emission. − Furthermore, rhodols were also used as fluorescent probes for inorganic (H 2 O 2 and HOCl) ( 5 and 6 ) or glutathione-detecting probes ( 7 ).…”

Human-T-Cell-Selective Fluorescent Probe

“…Emerging small molecule-based fluorescent probes have the potential to replace the antibodies. Recently, two fluorescent probes, CDgB and CDyB, for B cells’ distinction from T cells, have been developed by our group using unbiased screening with the Diversity-Oriented Fluorescence Library (DOFL), breaking the conventional method ( Figure 1 ) [ 10 , 11 ]. DOFL is a toolbox for cell-type distinction by combinatorial screening [ 12 , 13 ].…”