Cell labelling using a small fluorescent probe is an important technique in biomedical sciences. We previously developed a biocompatible and membrane‐permeable probe, CO‐1, which has low nonspecific binding affinity towards nontarget molecules. Although this background‐free tame probe has been utilized for labelling of various intracellular biomolecules in live cells, the probes’ backgroung‐free staining mechanism was not fully understood. Here, we propose that Gating‐Oriented Live‐cell Distinction (GOLD) mechanism occurs when ABCB1 transporter removes unbound CO‐1 molecules from mammalian cells and, in a minor role, DIRC2 pumps CO‐1 out from lysosomes. We also showed that solute carrier transporters were not involved in carrying CO‐1 inside of cells. The role of reporters in assisting the probes’ influx‐efflux was analyzed by the combination of CRISPR library sceenings and inhibitors test. In summary, tame probe CO‐1 cellular staining occurs in a dual mechanism where the probe moves freely through the cells membrane, but its washable property can be directly related to the action of ABCB1 transporter.
Human neutrophils are the most abundant leukocytes and have been considered as the first line of defence in the innate immune system. Selective imaging of live neutrophils will facilitate the in situ study of neutrophils in infection or inflammation events as well as clinical diagnosis. However, small‐molecule‐based probes for the discrimination of live neutrophils among different granulocytes in human blood have yet to be reported. Herein, we report the first fluorescent probe NeutropG for the specific distinction and imaging of active neutrophils. The selective staining mechanism of NeutropG is elucidated as metabolism‐oriented live‐cell distinction (MOLD) through lipid droplet biogenesis with the help of ACSL and DGAT. Finally, NeutropG is applied to accurately quantify neutrophil levels in fresh blood samples by showing a high correlation with the current clinical method.
Human neutrophils are the most abundant leukocytes and have been considered as the first line of defence in the innate immune system. Selective imaging of live neutrophils will facilitate the in situ study of neutrophils in infection or inflammation events as well as clinical diagnosis. However, small‐molecule‐based probes for the discrimination of live neutrophils among different granulocytes in human blood have yet to be reported. Herein, we report the first fluorescent probe NeutropG for the specific distinction and imaging of active neutrophils. The selective staining mechanism of NeutropG is elucidated as metabolism‐oriented live‐cell distinction (MOLD) through lipid droplet biogenesis with the help of ACSL and DGAT. Finally, NeutropG is applied to accurately quantify neutrophil levels in fresh blood samples by showing a high correlation with the current clinical method.
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