ABSTRACT
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.
Subject(s)
Fluorescent Dyes , Lysosomes , Animals , Biological Transport , Fluorescent Dyes/chemistry , Lysosomes/metabolism , Mammals/metabolism , Staining and LabelingABSTRACT
Stem cell research has gathered immense attention in the past decade due to the remarkable ability of stem cells for self-renewal and tissue-specific differentiation. Despite having numerous advancements in stem cell isolation and manipulation techniques, there is a need for highly reliable probes for the specific detection of live stem cells. Herein we developed a new fluorescence probe (CDy9) with high selectivity for mouse embryonic stem cells. CDy9 allows the detection and isolation of intact stem cells with marginal impact on their function and capabilities.