Access

You are not currently logged in.

Access your personal account or get JSTOR access through your library or other institution:

login

Log in to your personal account or through your institution.

Fura-2 Measurement of Cytosolic Free Ca2+ in Monolayers and Suspensions of Various Types of Animal Cells

Antonio Malgaroli, Daria Milani, Jacopo Meldolesi and Tullio Pozzan
The Journal of Cell Biology
Vol. 105, No. 5 (Nov., 1987), pp. 2145-2155
Stable URL: http://www.jstor.org/stable/1612643
Page Count: 11
  • More info
  • Cite this Item
Fura-2 Measurement of Cytosolic Free  Ca2+ in Monolayers and Suspensions of Various Types of Animal Cells
Preview not available

Abstract

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°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°C) temperature, no spots appeared, and cells remained diffusely labeled even after subsequent incubation at 32-37°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°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.

Page Thumbnails

  • Thumbnail: Page 
2145
    2145
  • Thumbnail: Page 
2146
    2146
  • Thumbnail: Page 
2147
    2147
  • Thumbnail: Page 
2148
    2148
  • Thumbnail: Page 
2149
    2149
  • Thumbnail: Page 
2150
    2150
  • Thumbnail: Page 
2151
    2151
  • Thumbnail: Page 
2152
    2152
  • Thumbnail: Page 
2153
    2153
  • Thumbnail: Page 
2154
    2154
  • Thumbnail: Page 
2155
    2155