Lysosomal pH Estimation via Fluorescent Dextrans

To estimate the relative lysosomal pH, subconfluent cells were loaded with 2.5 mg/mL pH-sensitive FITC coupled to 70 kDa dextran and 2.5 mg/mL pH-insensitive tetramethylrhodamine (TMR) coupled to 70 kDa dextran for 18 h, washed, and chased in fresh medium for 5 h. The medium was changed to Live Cell Imaging Solution (Thermo Fisher Scientific, Cat. A14291DJ) before image acquisition by LSM700 Confocal Laser Scanning Microscope (Zeiss). Images were analyzed using ImageJ software.

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Liu B., Chen R., Zhang Y., Huang J., Luo Y., Rosthøj S., Zhao C, & Jäättelä M. (2023). Cationic amphiphilic antihistamines inhibit STAT3 via Ca2+-dependent lysosomal H+ efflux. Cell Reports, 42(2), 112137.

Publication 2023

Live Cell Imaging Solution LSM700 Confocal Laser Scanning Microscope

Cell Confocal laser scanning microscope Dextran 70 Fitc Lysosomal Tetramethylrhodamine

Corresponding Organization : University of Copenhagen

Other organizations : Ocean University of China, BGI Group (China), Aarhus University

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Variable analysis

independent variables

Loading of cells with 2.5 mg/mL pH-sensitive FITC coupled to 70 kDa dextran
Loading of cells with 2.5 mg/mL pH-insensitive tetramethylrhodamine (TMR) coupled to 70 kDa dextran
Chase in fresh medium for 5 h

dependent variables

Relative lysosomal pH

control variables

Subconfluent cells
Loading time of 18 h
Live Cell Imaging Solution (Thermo Fisher Scientific, Cat. A14291DJ) for image acquisition
LSM700 Confocal Laser Scanning Microscope (Zeiss) for image acquisition
ImageJ software for image analysis

controls

Positive control: Not specified.
Negative control: Not specified.

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