Cell-permeable fluorescent indicator for cytosolic chloride

Area of Science:

• Cell biology
• Biochemistry
• Analytical chemistry

Background:

• Quinolinium-based fluorescent indicators for cytosolic chloride (Cl-) typically require invasive cell loading due to their high polarity and membrane impermeability.
• This limitation hinders real-time studies of cellular Cl- dynamics.

Purpose of the Study:

• To develop a novel approach for noninvasive loading of fluorescent indicators into living cells.
• To create a Cl(-)-sensitive fluorescent indicator that can be effectively trapped intracellularly.

Main Methods:

• A nonpolar precursor, 6-methoxy-N-ethyl-1,2-dihydroquinoline (diH-MEQ), was synthesized to mask the charged nitrogen.
• diH-MEQ was oxidized intracellularly to the fluorescent indicator 6-methoxy-N-ethylquinolium chloride (MEQ).
• Cell loading was achieved by incubating Swiss 3T3 fibroblasts and T84 cells with diH-MEQ, followed by a diH-MEQ-free buffer.

Main Results:

• MEQ exhibited fluorescence properties suitable for cellular imaging (344-nm absorbance, 440-nm emission, 0.70 quantum yield).
• MEQ fluorescence was quenched by Cl- via a collisional mechanism (Stern-Volmer constant, KCl = 145 M-1 in buffer, 19 M-1 in cells).
• The noninvasive loading method resulted in bright, uniform cell staining with minimal toxicity and good intracellular retention.

Conclusions:

• The novel diH-MEQ/MEQ system enables rapid, noninvasive loading of a Cl(-)-sensitive fluorescent indicator into living cells.
• This approach overcomes the limitations of previous quinolinium-based indicators, facilitating cellular Cl- studies.
• The indicator demonstrated good photophysical properties, low toxicity, and effective intracellular trapping.