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Simultaneous intracellular chloride and pH measurements using a GFP-based sensor

Nature Methods volume 7, pages 516–518 (2010)Cite this article

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Abstract

Chloride and protons perform important closely related roles in many cellular responses. Here we developed a ratiometric biosensor, ClopHensor, based on a highly chloride-sensitive Aequorea victoria GFP variant that is suited for the combined real-time optical detection of pH changes and chloride fluxes in live cells. We detected high chloride concentration in large dense-core exocytosis granules by targeting ClopHensor to these intracellular compartments.

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Figure 1: ClopHensor design and functional characterization.
Figure 2: ClopHensor pH and [Cl−]i calibration in live cells.
Figure 3: Measurement of chloride concentration in LDCVs.

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Acknowledgements

We thank P. Faraci for his assistance in preparing NPY-ClopHensor PC12 cell samples, S. Luin for his assistance with microscope analysis, S. Sulis-Sato and G. Lanza for their help with experimental setup, A. Cereseto for critical reading of the manuscript and G. Ratto for helpful discussions. The study was supported by Fondazione Monte dei Paschi di Siena (Monte dei Paschi Foundation) and by the Italian Ministry for University and Research (Fondo per gli Investimenti della Ricerca di Base RBLA03ER38).

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Author notes

  1. Fernanda Ricci and Laura Marchetti: These authors equally contributed to this work.

Authors and Affiliations

  1. National Enterprise for nanoScience and nanoTechnology, Istituto Nanoscienze, Consiglio Nazionale delle Ricerche and Scuola Normale Superiore, Pisa, Italy

    Daniele Arosio, Fernanda Ricci, Laura Marchetti, Roberta Gualdani, Lorenzo Albertazzi & Fabio Beltram

  2. Istituto di Biofisica, Consiglio Nazionale delle Ricerche, Trento Povo, Italy

    Daniele Arosio

  3. Italian Institute of Technology at National Enterprise for nanoScience and nanoTechnology, Center for Nanotechnology Innovation, Pisa, Italy

    Fernanda Ricci, Lorenzo Albertazzi & Fabio Beltram

Authors
  1. Daniele Arosio
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  2. Fernanda Ricci
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  3. Laura Marchetti
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  4. Roberta Gualdani
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  5. Lorenzo Albertazzi
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  6. Fabio Beltram
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Contributions

D.A. conceived the sensor, designed and performed experiments, analyzed data and wrote the paper; F.R. and L.M. generated ClopHensor constructs, and designed and performed experiments; R.G. performed sensor calibrations and kinetic analysis; L.A. performed experiments and analyzed data; F.B. interpreted the data, edited the paper and gave conceptual advice.

Corresponding author

Correspondence to Daniele Arosio.

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The authors declare no competing financial interests.

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Supplementary Figures 1–6, Supplementary Tables 1–2 and Supplementary Data (PDF 1559 kb)

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Arosio, D., Ricci, F., Marchetti, L. et al. Simultaneous intracellular chloride and pH measurements using a GFP-based sensor. Nat Methods 7, 516–518 (2010). https://doi.org/10.1038/nmeth.1471

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