Fluorescence analysis of the labile iron pool of mammalian cells
Fluorescence analysis of the labile iron pool of mammalian cells
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DOI：
10.1006/abio.1997.2126
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发表时间：
1997-05-15
影响因子：
2.9
通讯作者：
Cabantchik, ZI
中科院分区：
文献类型：
--
作者：
Epsztejn, S;Kakhlon, O;Cabantchik, ZI
细胞的不稳定铁池（LIP）构成了铁的胞浆部分，其可被渗透螯合剂接近并且包含细胞的代谢和催化活性铁，LIP通过来自细胞外和细胞内来源的铁的平衡运动来维持。该探针快速、化学计量和可逆地结合Fe（II），同时形成荧光猝灭的CA-Fe复合物。细胞通过其乙酰甲氧基前体CA-AM负载CA，达到1 - 10 μ M的细胞内浓度并保持完全活力。LIP在此操作性地定义为细胞的"游离"铁和CA结合铁的总和。（a）负载CA的细胞中CA的总细胞内浓度（[CA]（t）），其通过给定细胞悬浮液中CA的荧光测量、细胞数目和细胞体积来估计;（B）细胞内[CA-Fe]，与金属结合的[CA]的浓度（> 95%铁），其由通过添加高渗透性和高亲和力结合螯合剂如水杨醛-异烟酰-腙（SIH）引起的荧光的相对增加（Δ F）和[CA]（t）的值评估;和（c）"游离"细胞铁浓度[Fe（II）]，其由在悬浮液中生长的各种细胞系中CA-Fe（II）的解离常数（K-d）的实验测定值计算（K-d = 0.22 +/-0.01 μ M）。细胞LIP值定义为[CA-Fe]和[Fe]之和。它是由[CA]（t）和[CA-Fe]的实验测定和[CA-Fe]的K-d值应用质量定律方程计算得到的。静息红系和髓系细胞的LIP估计值在0.2 - 1.5 μ M范围内。该方法为在线监测正常和异常细胞铁供应条件下的胞浆铁和评估活细胞内铁的动态变化提供了一种简单、无创的工具。(C)北京：科学出版社.
The labile iron pool (LIP) of cells constitutes a cytosolic fraction of iron which is accessible to permeant chelators and contains the cells' metabolically and catalytically reactive iron, LIP is maintained by a balanced movement of iron from extra- and intracellular sources, We describe here an approach for tracing LIP levels in living cells based on the fluorescent probe calcein (CA). This probe binds Fe(II) rapidly, stoichiometrically, and reversibly while forming fluorescence-quenched CA-Fe complexes, Cells are loaded with CA via its acetomethoxy precursor CA-AM, attaining 1-10 mu M intracellular concentrations and retaining full viability, LIP is defined here operationally as the sum of ''free'' and CA-bound iron of the cell, The method for assessing LIP is based on the measurement of: (a) the total intracellular concentration of CA in CA-loaded cells ([CA](t)), which is estimated hom fluorimetric measurements of CA in a given suspension of cells, the number of cells, and the cell volume; (b) the intracellular [CA-Fe], the concentration of [CA] bound to metals (>95% iron), which is assessed from the relative rise in fluorescence (Delta F) elicited by addition of highly permeant and high-affinity binding chelators such as salicyladehyde-isonicotinoyl-hydrazone (SIH) and the value of [CA](t); and (c) the ''free'' cell iron concentration [Fe(II)], which is computed from the experimentally determined values of CA-Fe(II)'s dissociation constant (K-d) in various cell lines grown in suspension (K-d = 0.22 +/- 0.01 mu M). The value of cellular LIP is defined as the sum of [CA-Fe] and [Fe]. It is derived from the experimental determination of [CA](t) and [CA-Fe] and from calculation of [Fe] by application of the mass law equation using the K-d value of [CA-Fe], The estimated values of LIP for resting erythroid and myeloid cells are in the range of 0.2-1.5 mu M. The values varied commensurately with cell iron loads and iron chelator treatment, The method provides a simple, noninvasive tool for on-line monitoring of cytosolic iron under normal and abnormal conditions of cell iron supply and for assessing the dynamics of intracellular iron in living cells. (C) 1997 Academic Press.