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
- 34 Downloads
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 Words1,6-Diphenyl-1,3,5-hexatriene (DPH) living cells phosphatidylcholine phase fluorometry steady-state fluorescence
Unable to display preview. Download preview PDF.
- 1.W. L. Bergman et al., (1984),Biophys. Biochim. Acta 772, 328–336.Google Scholar
- 5.D. Grunberger et al. (1982)Biophys. Biochim. Acta 688, 764–774.Google Scholar
- 6.C. G. Morgan et al. (1982)Biophys. Biochim. Acta 692, 196–201.Google Scholar
- 7.G. Morrot et al. (1988)Biochemistry 28, 3456–3462.Google Scholar
- 10.M. Shinitzky (1978)Biophys. Biochim. Acta 515, 367–394.Google Scholar
- 11.C. Szczepaniaket al. (1991)IVème Réunion du Groupe d'Etude des Interactions Molécules Membranes (GEIMM), Strasbourg, Nov. 20–22, abstract.Google Scholar
- 12.A. J. Verkleij et al. (1973)Biophys. Biochim. Acta 323, 178–193.Google Scholar