Ratiometric fluorescence imaging of cell surface pH by poly(ethylene glycol)-phospholipid conjugated with fluorescein isothiocyanate

Abstract

Various physiological and pathological processes are accompanied with the alteration of pH at extracellular juxtamembrane region. Accordingly, the methods to analyze the cell surface pH have been demanded in biological and medical sciences. In this study, we have established a novel methodology for cell surface pH imaging using poly(ethylene glycol)-phospholipid (PEG-lipid) as a core structure of ratiometric fluorescent probes. PEG-lipid is a synthetic amphiphilic polymer originally developed for the cell surface modification in transplantation therapy. Via its hydrophobic alkyl chains of the phospholipid moiety, PEG-lipid is, when applied extracellularly, spontaneously inserted into the plasma membrane and retained at the surface of the cells. We have demonstrated that the PEG-lipid conjugated with fluorescein isothiocyanate (FITC-PEG-lipid) can be used as a sensitive and reversible cell-surface-anchored pH probe between weakly alkaline and acidic pH with an excellent spatiotemporal resolution. The remarkably simple procedure for cell-surface labeling with FITC-PEG-lipid would also be advantageous when considering its application to high-throughput in vitro assay. This study further indicates that various probes useful for the investigation of juxtamembrane environments could also be developed by using PEG-lipid as the core structure for bio-membrane anchoring.

Dynamic Extracellular Imaging of Biochemical Cell Activity Using InGaN/GaN Nanowire Arrays as Nanophotonic Probes

“…Recently, ratiometric fluorescence sensors were demonstrated to monitor the extracellular pH with a simultaneous high spatiotemporal resolution . However, due to stability issues such probes are applicable only in weak alkaline or acidic environments and the achievable pH resolution of ΔpH = 0.1 is significantly lower than the pH resolution demonstrated for InGaN/GaN NWHs here (ΔpH = 0.03). Optical cell imaging based on surface‐enhanced Raman scattering showed a subendosomal spatial resolution, but very limited temporal resolution still and a pH resolution of only ΔpH = 0.2 …”

Modification of human MSC surface with oligopeptide‐PEG‐lipids for selective binding to activated endothelium

“…Since we have already found that the incorporation of PEGlipid into cell surface reached the plateau at around 30 min, 17 1.2 × 10 8 molecules and 3.7 × 10 8 molecules on CCRF-CEM and hMSC, respectively, in case of 1 kDa PEG (Fig. 3).…”

“…Since we have already found that the incorporation of PEGlipid into cell surface reached the plateau at around 30 min, 17 the incubation time was fixed to be 30 min, and FITC-ES-bp(C)-PEG-lipid was used for CCRF-CEM and hMSC with different added concentration. The immobilized number of ES-bp onto cells depends on the concentration, and the analysis showed that the number of immobilized oligopeptides increases with the added concentration and (Fig.…”

A Novel Visible Range FRET Probe for Monitoring Acid Sphingomyelinase Activity in Living Cells

“…From the recorded graphs using the probe 5 a it was clearly visible, that the absolute fluorescence increase was the largest at pH 5.5. This behavior is easily explained by the fact that the fluorescence of fluorescein is increasing with higher pH . However, direct comparison showed that initial slopes decreased with higher pH and that the curve at pH 4.5 entered a plateau phase, while the curves at higher pH values were still increasing, clearly demonstrating a pH optimum below 5.0 under these conditions (Figure D).…”