Analytical Chemistry Insights
Synopsis: An open access, peer reviewed electronic journal that covers developments in the field including analytical methodology, techniques and instrumentation in the fundamental and applied areas of the field.
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Determination of the Labile Iron Pool of Human Lymphocytes using the Fluorescent Probe, CP655
Authors: Yongmin Ma, Zudong Liu, Robert C Hider and Frank Petrat
Publication Date: 17 Sep 2007
Analytical Chemistry Insights 2007:2 61-67
Yongmin Ma1, Zudong Liu1, Robert C Hider1 and Frank Petrat2
1Department of Pharmacy, King’s College London, Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NH, U.K. 2Institut für Physiologische Chemie, Universitätsklinikum, Hufelandstr. 55, D-45122 Essen,Germany.
Abstract: The present study introduces a method for determining the labile iron pool (LIP) in human lymphocytes. It is measured using the probe CP655, the fluorescence of which is stoichiometrically quenched by the addition of iron. The intracellular CP655 fluorescence in lymphocytes was quenched by increasing intracellular iron concentrations using the highly lipophilic 8-hydroxyquinoline iron complex. Intracellular fluorescence quenching, mediated by the physiological intracellular labile iron, can be recovered on the addition of excess membrane-permeable iron chelator, CP94. The intracellular probe concentration was measured using laser scanning microscopy. An ex situ calibration was performed in a “cytosolic” medium based on the determined intracellular CP655 concentration and probe fluorescence quenching in the presence of iron. The concentration of the LIP of healthy human lymphocytes was determined to be 0.57 ± 0.27 μM. The use of the fluorescent probe CP655 renders it possible to record the time course of iron uptake and iron chelation by CP94 in single intact lymphocytes.
Categories: Analytical chemistry
Keywords: lymphocytes; intracellular chelatable iron; fluorescence microscopy; fluorescent probe
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