Acidification of the extracellular juxtamembrane space is involved in various biological processes. However, imaging probes to investigate the spatio-temporal dynamics as well as the functional significance of cell surface pH are still limited. We established a method of cell surface pH imaging by using a membrane-anchored probe, poly(ethylene glycol)-phospholipid conjugated with fluorescein isothiocyanate (FITC-PEG-lipid). When added into the cell culture medium, FITC-PEG-lipid was inserted into the plasma membrane via its phospholipid moiety. FITC-PEG-lipid was retained at cell surface. The ratiometric readout of fluorescence was reversible and unique to the extracellular pH in the range of neutral and weakly acidic pH. The measurement with FITC-PEG-lipid was accurate enough to distinguish the difference of 0.1 pH unit near the inflection point of fluorescence ratio. Our study demonstrates that FITC-PEG-lipid is useful as a cell surface-anchored pH probe. The simple labeling procedure is advantageous especially when considering its application to high-throughput in vitro assay. Furthermore, PEG-lipid holds a great potential as the membrane anchor of various analytical probes to investigate the juxtamembrane environments.

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