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Journal of Fluorescence

, Volume 3, Issue 4, pp 251–255 | Cite as

Interaction of a phosphatidylcholine derivative of 1,6-diphenyl-1,3,5-hexatriene (DPH) with intact living cells: Steady-state fluorescence polarization and phase fluorometry studies

  • L. Miccoli
  • C. Szczepaniak
  • D. Dumas
  • S. Savonnière
  • S. Muller
  • M. C. Carré
  • M. Donner
Fluorescence of Membranes

Abstract

The potential interest of DPH-PC was checked with a macrophagic cell line (P388D1). The uptake of DPH-PC was associated with a rapid increase in both fluorescence intensity and a slow decrease in anisotropy values. A flow cytometry comparative study with DPH revealed in both cases the existence of two cell subpopulations with different labeling levels. The analysis of fluorescence decay of DPH-PC showed two components. The fractional intensity of the main component (9.7 ns) is higher than 92%. The Lorentzian distribution of the main lifetime presents an important homogeneity. The observation that an increase in temperature induced a decrease in steady state anisotropy values but did not affect the lifetime suggests that the anisotropy variations effectively reflect modifications in the cohesion of probe micro-surroundings. A transmembrane diffusional phenomenon of a fraction of fluorescent phospholipids (205) was suggested by a study with a nonpermeant membrane quencher. The transmembrane diffusion was confirmed by extraction of the phospholipid analog with fatty acid free BSA. The use of inhibitors of endogenous phospholipase A2 showed a progressive hydrolysis of the fluorescent phospholipid. Nevertheless, the hydrolysis can be neglected in the case of short term interactions with cells (<30 min). Therefore, it can be assumed that DPH-PC can be used as a membrane probe.

Key Words

1,6-Diphenyl-1,3,5-hexatriene (DPH) living cells phosphatidylcholine phase fluorometry steady-state fluorescence 

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Copyright information

© Plenum Publishing Corporation 1993

Authors and Affiliations

  • L. Miccoli
    • 1
  • C. Szczepaniak
    • 1
  • D. Dumas
    • 2
  • S. Savonnière
    • 1
  • S. Muller
    • 1
  • M. C. Carré
    • 1
  • M. Donner
    • 1
  1. 1.Groupe de Recherches sur les Interactions Moléculaires aux InterfacesINSERM-CO 10Vandoeuvre les NancyFrance
  2. 2.Laporatoire d'Hémorhéologie-HématologieFaculté de MédecineVandoeuvre les NancyFrance

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