Nucleoplasmic and cytoplasmic differences in the fluorescence properties of the calcium indicator Fluo-3

Carmen Perez-Terzic, Lisa Stehno-Bittel, David E. Clapham

Research output: Contribution to journalArticle

87 Scopus citations

Abstract

The fluorescent indicator Fluo-3 is widely used to monitor the calcium concentration ([Ca2+) in the cytoplasm and nucleus of various cells. Estimates of nuclear [Ca2+] are based on the assumption of identical behavior of Fluo-3 in different cellular compartments. The assumption is not valid if the fluorescence properties of the dye are altered by the nuclear environment, independent of the [Ca2+]. To determine the effects of the nucleoplasm on the behavior of Fluo-9, we applied laser scanning confocal microscopy and spectrophotometry to measure fluorescence intensity as well as emission and absorbance spectra of the Ca2+ indicator, Fluo-3. Spectra were measured in intact Xenopus oocytes, neuroblastoma cells, and cytoplasmic and nucleoplasmic homogenates. The fluorescence signal in intact cells loaded with Fluo-3 was approximately 2-times higher in the nucleus when compared to the cytoplasm. The fluorescence intensity of Fluo-3 in nucleoplasmic homogenates was higher than in cytoplasmic homogenates or internal buffers even when [Ca2+] was clamped. Despite identical [Ca2+], pH, and temperature, the emission and absorbance spectra of Fluo-3 from nuclear homogenates displayed a higher fluorescence at each wavelength measured when compared to spectra from cytoplasmic homogenates or internal buffer solutions, and saturated above 100 nM. These findings demonstrate that the composition of the nucleoplasm changes the fluorescence properties of the calcium indicator Fluo-3. Consequently, analysis of nuclear calcium dynamics must take into account the distinct behavior of Fluo-3 in different cellular compartments.

Original languageEnglish (US)
Pages (from-to)275-282
Number of pages8
JournalCell Calcium
Volume21
Issue number4
DOIs
StatePublished - Apr 1997

ASJC Scopus subject areas

  • Physiology
  • Molecular Biology
  • Cell Biology

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