Abstract
Use of Fura-2 in whole cell suspensions to measure changes in intracellular Ca2+ is probably one of the simplest, yet most widely used protocols described in this volume. Whole cell suspensions are loaded with Fura-2 and then placed into a cuvette-based fluorimetric system (measuring 510 nm emission at alternating 340/340 nm excitation). Cells can be stimulated with agonists and antagonists to enable temporal response profiling and concentration–response curves to be constructed. The protocol can be used for a wide range of cells including those transfected with Ca2+-signaling proteins, e.g., receptors and channels. Loading characteristics and the need for agents to retain loaded dye (e.g., probenecid) need to be determined empirically. Calibration of whole cell suspensions to convert the fluorescent signal into Ca2+ is simply performed using Triton-X lysis (to determine R max) and EGTA chelation (to determine R min).
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Clapham D (1995) Calcium signalling. Cell 80:259–268
Berridge MJ (1993) Inositol trisphosphate and Ca2+ signalling. Nature 361:315–325
Grynkiewicz G, Poenie M, Tsien RY (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260:3440–3449
Lambert DG, Wojcikiewicz RJH, Safrany ST, Whitham EM, Nahorski SR (1992) Muscarinic receptors, phosphoinositide metabolism and intracellular calcium in neuronal cells. Prog Neuropsychopharmacol Biol Psychiatry 16:253–270
Cobbold PH, Rink RJ (1987) Fluorescence and bioluminescence measurement of cytoplasmic free calcium. Biochem J 248:313–328
Hirota K, Lambert DG (1997) A comparative study of L-type voltage sensitive Ca2+ channels in rat brain regions and cultured neuronal cells. Neurosci Lett 223:169–172
Brezden CB, Hedley DW, Rauth AM (1994) Constitutive expression of P-glycoprotein as a determinant of loading with fluorescent calcium probes. Cytometry 17:343–348
Edelman JL, Kajimura M, Woldemussie E, Sachs G (1994) Differential effects of carbachol on calcium entry and release in CHO cells expressing the m3 muscarinic receptor. Cell Calcium 16:181–193
Iredale PA, Hill SJ (1993) Increases in intracellular calcium via activation of an endogenous P(2)-purinoceptor in cultured CHO-k1 cells. Br J Pharmacol 110:1305–1310
McDonald J, Batuwangala M, Lambert DG (2007) Role of urotensin II and its receptor in health and disease. J Anesth 3:378–389
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Patel, A., Hirst, R.A., Harrison, C., Hirota, K., Lambert, D.G. (2013). Measurement of [Ca2+]i in Whole Cell Suspensions Using Fura-2. In: Lambert, D., Rainbow, R. (eds) Calcium Signaling Protocols. Methods in Molecular Biology, vol 937. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-086-1_2
Download citation
DOI: https://doi.org/10.1007/978-1-62703-086-1_2
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-085-4
Online ISBN: 978-1-62703-086-1
eBook Packages: Springer Protocols