Petrat F, de Groot H, Rauen U
Institut für Physiologische Chemie, Universitätsklinikum, Hufelandstr. 55, Essen, D-45122, Germany.
Arch Biochem Biophys. 2000 Apr 1;376(1):74-81. doi: 10.1006/abbi.2000.1711.
We have previously established a method of detecting intracellular chelatable iron in viable cells based on digital fluorescence microscopy. To quantify cellular chelatable iron, it was crucial to determine the intracellular indicator concentration. In the present study, we therefore adapted the method to confocal laser scanning microscopy, which should allow the determination of the indicator concentration on the single-cell level. The fluorescent heavy-metal indicator phen green SK (PG SK), the fluorescence of which is quenched by iron, was loaded into cultured rat hepatocytes. The hepatocellular fluorescence increased when cellular chelatable iron available to PG SK was removed from the probe by an excess of the membrane-permeable transition metal chelator 2,2'-dipyridyl (2, 2'-DPD, 5 mM). We optimized the scanning parameters for quantitatively recording changes in fluorescence and determined individual intracellular PG SK concentrations from the unquenched cellular fluorescence (after 2,2'-DPD) compared with PG SK standards in a "cytosolic" medium. An ex situ calibration method based on laser scanning microscopy was set up to determine the concentration of cellular chelatable iron from the increase of PG SK fluorescence after addition of 2,2'-DPD (5 mM). As the stoichiometry of the PG SK:Fe(2+) complex was 3:1 as long as PG SK was not limiting, cellular chelatable iron was calculated directly from absolute changes in cellular fluorescence. Using this method, we found 2.5 +/- 2.2 microM chelatable iron in hepatocytes. This method makes it possible to determine the pool of chelatable iron in single vital cells independently of cellular differences (e.g., dye loading, cell volume) in heterogeneous cell populations.
我们之前基于数字荧光显微镜建立了一种检测活细胞内可螯合铁的方法。为了量化细胞可螯合铁，确定细胞内指示剂浓度至关重要。因此，在本研究中，我们将该方法应用于共聚焦激光扫描显微镜，这应该能够在单细胞水平上确定指示剂浓度。将荧光重金属指示剂菲咯绿SK（PG SK）（其荧光会被铁淬灭）加载到培养的大鼠肝细胞中。当过量的膜通透性过渡金属螯合剂2,2'-联吡啶（2,2'-DPD，5 mM）从探针中去除可供PG SK利用的细胞可螯合铁时，肝细胞荧光增强。我们优化了用于定量记录荧光变化的扫描参数，并根据未淬灭的细胞荧光（2,2'-DPD处理后）与“胞质”培养基中的PG SK标准品相比，确定了单个细胞内PG SK的浓度。建立了一种基于激光扫描显微镜的异位校准方法，以根据添加2,2'-DPD（5 mM）后PG SK荧光的增加来确定细胞可螯合铁的浓度。由于只要PG SK不成为限制因素，PG SK:Fe(2+)复合物的化学计量比为3:1，因此可直接根据细胞荧光的绝对变化计算细胞可螯合铁。使用该方法，我们在肝细胞中发现了2.5±2.2 microM的可螯合铁。该方法使得能够独立于异质细胞群体中的细胞差异（例如染料加载、细胞体积）来确定单个活细胞中的可螯合铁池。
