We previously developed a hydrolase-based fluorescence amplification method for antigen-specific cell labelling, in which fluorescent substrates stained cells by a non-covalent hydrophobic interaction. To improve the substrates retention in cells, we examined the effect of a chloroacetyl group modification on the substrate retention. We found that the chloroacetyl group suppressed the dissociation of the substrate after forming a covalent bond with intracellular proteins. However’ the slow reaction speed of the chloroacetyl group allowed dissociation for cells in the early stage of the staining reaction.
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