The fluorescent indicator fura-2 has been applied to a variety of cell types in order to set up appropriate conditions for measurements of the cytosolic concentration of free ionized Ca2+ [( Ca2+]i) in both cell suspensions and single cells analyzed in a conventional fluorimeter or in a fluorescence microscope equipped for quantitative analyses (with or without computerized image analyses), respectively. When the usual procedure for fluorescence dye loading (i.e., incubation at 37 degrees C with fura-2 acetoxy-methyl ester) was used, cells often exhibited a nonhomogeneous distribution of the dye, with marked concentration in multiple small spots located preferentially in the perinuclear area. These spots (studied in detail in human skin fibroblasts), were much more frequent in attached than in suspended cells, and were due to the accumulation (most probably by endocytosis) of the dye within acidic organelles after hydrolysis by lysosomal enzyme(s). When loading with fura-2 was performed at low (15 degrees C) temperature, no spots appeared, and cells remained diffusely labeled even after subsequent incubation at 32-37 degrees C for up to 2 h. Homogeneous distribution of the dye is a prerequisite for appropriate [Ca2+]i measurement. In fact, comparison of the results obtained in human skin fibroblasts labeled at either 37 or 15 degrees C demonstrated in spotty cells a marked apparent blunting of Ca2+ transients evoked by application of bradykinin. Additional problems were encountered when using fura-2. Leakage of the dye from loaded cells to the extracellular medium markedly affected the measurements in cell suspensions. This phenomenon was found to depend on the cell type, and to markedly decrease when temperature was lowered, suggesting the involvement of a facilitated transport. Calibration of fluorescence signals in terms of absolute [Ca2+]i was complicated by the increased fluorescence of fura-2 in the intracellular environment. To solve this problem we propose an in situ calibration procedure based on measurements carried out on cells in which [Ca2+]i was massively lowered (by loading the probe in a Ca2+-free medium) or increased (by treatment with the Ca2+ ionophore ionomycin, applied in a medium containing 3 mM Ca2+). These results provide explanations and, at least partial, solutions to the major problems encountered when using fura-2, and should thus be of help in clarifying the proper usage of the dye in [Ca2+]i measurements.
Skip Nav Destination
Article navigation
1 November 1987
Article|
November 01 1987
Fura-2 measurement of cytosolic free Ca2+ in monolayers and suspensions of various types of animal cells.
A Malgaroli,
A Malgaroli
Department of Pharmacology, University of Milan, Italy.
Search for other works by this author on:
D Milani,
D Milani
Department of Pharmacology, University of Milan, Italy.
Search for other works by this author on:
J Meldolesi,
J Meldolesi
Department of Pharmacology, University of Milan, Italy.
Search for other works by this author on:
T Pozzan
T Pozzan
Department of Pharmacology, University of Milan, Italy.
Search for other works by this author on:
A Malgaroli
Department of Pharmacology, University of Milan, Italy.
D Milani
Department of Pharmacology, University of Milan, Italy.
J Meldolesi
Department of Pharmacology, University of Milan, Italy.
T Pozzan
Department of Pharmacology, University of Milan, Italy.
Online ISSN: 1540-8140
Print ISSN: 0021-9525
J Cell Biol (1987) 105 (5): 2145–2155.
Citation
A Malgaroli, D Milani, J Meldolesi, T Pozzan; Fura-2 measurement of cytosolic free Ca2+ in monolayers and suspensions of various types of animal cells.. J Cell Biol 1 November 1987; 105 (5): 2145–2155. doi: https://doi.org/10.1083/jcb.105.5.2145
Download citation file:
Sign in
Don't already have an account? Register
Client Account
You could not be signed in. Please check your email address / username and password and try again.
Could not validate captcha. Please try again.
Sign in via your Institution
Sign in via your InstitutionSuggested Content
Email alerts
Advertisement
Advertisement