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Intracellular Calcium Recording After Purinoceptor Activation Using a Video-Microscopy Equipment

  • Maria Teresa Miras-Portugal
  • Felipe Ortega
  • Javier Gualix
  • Raquel Perez-Sen
  • Esmerilda G. Delicado
  • Rosa Gomez-VillafuertesEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2041)

Abstract

Calcium is one of the most important intracellular messengers, triggering a wide range of cellular responses. Changes in intracellular free calcium concentration can be measured using calcium sensitive fluorescent dyes, which are either EGTA- or BAPTA-based organic molecules that change their spectral properties in response to Ca2+ binding. One of the most common calcium indicators is the ratiometric dye Fura-2. The main advantage of using ratiometric dyes is that the ratio signal is independent of the illumination intensity, dye concentration, photobleaching, and focus changes among others, allowing for the concentration of intracellular calcium to be determined independently of these artifacts. In this protocol, we describe the use of Fura-2 to measure intracellular calcium elevations in single cultured cells after purinoceptor activation using a video-microscopy equipment. This method, usually known as calcium imaging, allows for real-time quantification of intracellular calcium dynamics and can be adapted to measure agonist mediated intracellular calcium responses due to the activation of different purinergic receptors in several cellular models using the appropriate growth conditions.

Key words

Calcium imaging Fura-2 Ratiometric calcium dye Purinergic receptor Intracellular free calcium concentration Video microscopy Calcium responses 

Notes

Acknowledgments

This work was supported by Ministerio de Economia y Competitividad (MINECO, BFU 2014-53654-P), Red de Excelencia Consolider-Ingenio Spanish Ion Channel Initiative (BFU2015-70067REDC), Comunidad de Madrid (BRADE-CM S2013/ICE-2958), and Fundación Ramón Areces (PR2018/16-02). F. Ortega is the recipient of a Ramón y Cajal contract (RYC-2013-13290).

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Maria Teresa Miras-Portugal
    • 1
    • 2
    • 3
  • Felipe Ortega
    • 1
    • 2
    • 3
  • Javier Gualix
    • 1
    • 2
    • 3
  • Raquel Perez-Sen
    • 1
    • 2
    • 3
  • Esmerilda G. Delicado
    • 1
    • 2
    • 3
  • Rosa Gomez-Villafuertes
    • 1
    • 2
    • 3
    Email author
  1. 1.Departamento de Bioquímica y Biología Molecular, Facultad de VeterinariaUniversidad Complutense de MadridMadridSpain
  2. 2.Instituto Universitario de Investigación en Neuroquímica (IUIN)MadridSpain
  3. 3.Instituto de Investigación Sanitaria del Hospital Clínico San Carlos (IdISSC)MadridSpain

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