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.
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References
Abbracchio MP, Burnstock G (1994) Purinoceptors: are there families of P2X and P2Y purinoceptors? Pharmacol Ther 64(3):445–475
Burnstock G (2007) Purine and pyrimidine receptors. Cell Mol Life Sci 64(12):1471–1483. https://doi.org/10.1007/s00018-007-6497-0
Berridge MJ, Bootman MD, Roderick HL (2003) Calcium signalling: dynamics, homeostasis and remodelling. Nat Rev Mol Cell Biol 4(7):517–529. https://doi.org/10.1038/nrm1155
Petersen OH, Michalak M, Verkhratsky A (2005) Calcium signalling: past, present and future. Cell Calcium 38(3–4):161–169. https://doi.org/10.1016/j.ceca.2005.06.023
Mateo J, Garcia-Lecea M, Miras-Portugal MT, Castro E (1998) Ca2+ signals mediated by P2X-type purinoceptors in cultured cerebellar Purkinje cells. J Neurosci 18(5):1704–1712
Garcia-Lecea M, Delicado EG, Miras-Portugal MT, Castro E (1999) P2X2 characteristics of the ATP receptor coupled to [Ca2+]i increases in cultured Purkinje neurons from neonatal rat cerebellum. Neuropharmacology 38(5):699–706
Hervas C, Perez-Sen R, Miras-Portugal MT (2003) Coexpression of functional P2X and P2Y nucleotide receptors in single cerebellar granule cells. J Neurosci Res 73(3):384–399. https://doi.org/10.1002/jnr.10676
Nobile M, Monaldi I, Alloisio S, Cugnoli C, Ferroni S (2003) ATP-induced, sustained calcium signalling in cultured rat cortical astrocytes: evidence for a non-capacitative, P2X7-like-mediated calcium entry. FEBS Lett 538(1–3):71–76
Carrasquero LM, Delicado EG, Bustillo D, Gutierrez-Martin Y, Artalejo AR, Miras-Portugal MT (2009) P2X7 and P2Y13 purinergic receptors mediate intracellular calcium responses to BzATP in rat cerebellar astrocytes. J Neurochem 110(3):879–889. https://doi.org/10.1111/j.1471-4159.2009.06179.x
Gomez-Villafuertes R, del Puerto A, Diaz-Hernandez M, Bustillo D, Diaz-Hernandez JI, Huerta PG, Artalejo AR, Garrido JJ, Miras-Portugal MT (2009) Ca2+/calmodulin-dependent kinase II signalling cascade mediates P2X7 receptor-dependent inhibition of neuritogenesis in neuroblastoma cells. FEBS J 276(18):5307–5325. https://doi.org/10.1111/j.1742-4658.2009.07228.x
Traves PG, Pimentel-Santillana M, Carrasquero LM, Perez-Sen R, Delicado EG, Luque A, Izquierdo M, Martin-Sanz P, Miras-Portugal MT, Bosca L (2013) Selective impairment of P2Y signaling by prostaglandin E2 in macrophages: implications for Ca2+-dependent responses. J Immunol 190(8):4226–4235. https://doi.org/10.4049/jimmunol.1203029
Gomez-Villafuertes R, Rodriguez-Jimenez FJ, Alastrue-Agudo A, Stojkovic M, Miras-Portugal MT, Moreno-Manzano V (2015) Purinergic receptors in spinal cord-derived ependymal stem/progenitor cells and their potential role in cell-based therapy for spinal cord injury. Cell Transplant 24(8):1493–1509. https://doi.org/10.3727/096368914X682828
Grynkiewicz G, Poenie M, Tsien RY (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260(6):3440–3450
Tsien RY, Rink TJ, Poenie M (1985) Measurement of cytosolic free Ca2+ in individual small cells using fluorescence microscopy with dual excitation wavelengths. Cell Calcium 6(1–2):145–157
Sanchez-Nogueiro J, Marin-Garcia P, Leon D, Leon-Otegui M, Salas E, Gomez-Villafuertes R, Gualix J, Miras-Portugal MT (2009) Axodendritic fibres of mouse cerebellar granule neurons exhibit a diversity of functional P2X receptors. Neurochem Int 55(7):671–682. https://doi.org/10.1016/j.neuint.2009.06.009
Lattanzio FA Jr, Bartschat DK (1991) The effect of pH on rate constants, ion selectivity and thermodynamic properties of fluorescent calcium and magnesium indicators. Biochem Biophys Res Commun 177(1):184–191
Petr MJ, Wurster RD (1997) Determination of in situ dissociation constant for Fura-2 and quantitation of background fluorescence in astrocyte cell line U373-MG. Cell Calcium 21(3):233–240
Di Garbo A, Alloisio S, Nobile M (2012) P2X7 receptor-mediated calcium dynamics in HEK293 cells: experimental characterization and modelling approach. Phys Biol 9(2):026001. https://doi.org/10.1088/1478-3975/9/2/026001
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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Miras-Portugal, M.T., Ortega, F., Gualix, J., Perez-Sen, R., Delicado, E.G., Gomez-Villafuertes, R. (2020). Intracellular Calcium Recording After Purinoceptor Activation Using a Video-Microscopy Equipment. In: Pelegrín, P. (eds) Purinergic Signaling. Methods in Molecular Biology, vol 2041. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9717-6_23
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DOI: https://doi.org/10.1007/978-1-4939-9717-6_23
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