Deka C, Lehnert B E, Lehnert N M, Jones G M, Sklar L A, Steinkamp J A
Life Sciences Division, Los Alamos National Laboratory, New Mexico, USA.
Cytometry. 1996 Nov 1;25(3):271-9. doi: 10.1002/(SICI)1097-0320(19961101)25:3<271::AID-CYTO8>3.0.CO;2-I.
Fluorescent antibodies are often used to measure the number of receptor sites on cells. The quantitative estimate of the number of receptor sites using this procedure assumes that the fluorescence intensity on a cell is proportional to the number of bound antibodies. Quenching may invalidate this assumption. For many fluorophores, intermolecular interactions and energy transfer between molecules in close proximity to one another results in self-quenching. This effect can occur in antibody probes with a high fluorochrome to protein (F/P) ratio. It can also occur due to close proximity antibodies relative to one another on a highly labeled cell surface. Since self-quenching is accompanied by a change in the fluorescence decay and a decrease in the fluorescence lifetime, it may be conveniently identified using fluorescence lifetime spectroscopy. In this paper we apply the phase-sensitive detection method to investigate the impact of self-quenching on fluorescence lifetimes by flow cytometry, using a model system consisting of FITC conjugated anti-mouse Thy1.2 antibodies bound to murine thymus cells. We show that in addition to the expected variation of lifetimes as a function of F/P ratio of the probes, the fluorescence lifetime diminishes also as a function of antibody labeling concentration on the cell surface. This is consistent with self-quenching effects expected at high densities of FITC molecules.
荧光抗体常被用于测量细胞上受体位点的数量。使用该方法对受体位点数量进行定量估计时，假定细胞上的荧光强度与结合抗体的数量成正比。淬灭可能会使这一假设无效。对于许多荧光团而言，彼此靠近的分子间的分子间相互作用和能量转移会导致自淬灭。这种效应可能发生在荧光染料与蛋白质（F/P）比例较高的抗体探针中。在高度标记的细胞表面，由于抗体彼此靠近也可能发生这种效应。由于自淬灭伴随着荧光衰减的变化和荧光寿命的缩短，使用荧光寿命光谱法可以方便地识别它。在本文中，我们应用相敏检测方法，通过流式细胞术研究自淬灭对荧光寿命的影响，使用的模型系统由与小鼠胸腺细胞结合的异硫氰酸荧光素（FITC）偶联的抗小鼠Thy1.2抗体组成。我们表明，除了预期的寿命随探针F/P比例的变化外，荧光寿命还会随着细胞表面抗体标记浓度的变化而缩短。这与在高密度FITC分子下预期的自淬灭效应一致。
