Steinkamp J A, Lehnert B E, Lehnert N M
Life Sciences Division, Los Alamos National Laboratory, NM 87545, USA.
J Immunol Methods. 1999 Jun 24;226(1-2):59-70. doi: 10.1016/s0022-1759(99)00053-8.
We report a flow cytometric fluorescence lifetime-based method to discriminate damaged/dead from viable cells in immunofluorescently labeled populations using propidium iodide as a dye-exclusion viability probe. Fluorescence signals from propidium iodide and the anti-thymus cell-surface immunofluorescence marker fluorochromes, phycoerythrin and phycoerythrin/Texas Red (tandem conjugate), which have overlapping emission spectra with propidium iodide, are resolved based on differences in their fluorescence emission lifetimes using phase-sensitive detection. Mouse thymus cell samples were first labeled separately with anti-Thy 1.2 antibody directly conjugated to phycoerythrin and to phycoerythrin/Texas Red and propidium iodide. Labeled cells were then analyzed to determine the lifetimes of the immunofluorescence markers and propidium iodide. Based on these results, rat and mouse thymocytes labeled with anti-Thy 1.1 conjugated to phycoerythrin and anti-Thy 1.2 conjugated to phycoerythrin/Texas Red, respectively, were suspended in phosphate buffered saline containing propidium iodide, and were analyzed as they passed through a flow chamber and crossed a high-frequency, intensity-modulated (sinusoidal) laser excitation beam. The resulting immunofluorescence and propidium iodide signals were resolved based on differences in fluorescence lifetimes expressed as phase shifts using phase-sensitive detection and displayed as frequency distribution histograms and bivariate contour diagrams. This technology provides a new method to resolve immunofluorescence and propidium iodide signals from overlapping fluorescence emission spectra and a flow cytometric lifetime-based technique to quantify damaged/dead cells in immunofluorescence studies.
我们报告了一种基于流式细胞术荧光寿命的方法，该方法使用碘化丙啶作为染料排除活力探针，在免疫荧光标记的细胞群体中区分受损/死亡细胞与活细胞。碘化丙啶以及抗胸腺细胞表面免疫荧光标记荧光染料（藻红蛋白和藻红蛋白/德克萨斯红（串联共轭物），其发射光谱与碘化丙啶重叠）发出的荧光信号，通过相敏检测基于它们荧光发射寿命的差异进行分辨。小鼠胸腺细胞样本首先分别用直接与藻红蛋白以及藻红蛋白/德克萨斯红共轭的抗Thy-1.2抗体和碘化丙啶进行标记。然后对标记的细胞进行分析，以确定免疫荧光标记和碘化丙啶的寿命。基于这些结果，分别用与藻红蛋白共轭的抗Thy-1.1和与藻红蛋白/德克萨斯红共轭的抗Thy-1.2标记的大鼠和小鼠胸腺细胞，悬浮于含有碘化丙啶的磷酸盐缓冲盐溶液中，并在它们通过流动腔并穿过高频、强度调制（正弦）激光激发束时进行分析。通过相敏检测，根据以相移表示的荧光寿命差异分辨得到的免疫荧光和碘化丙啶信号，并显示为频率分布直方图和双变量等高线图。该技术提供了一种从重叠荧光发射光谱中分辨免疫荧光和碘化丙啶信号的新方法，以及一种基于流式细胞术寿命的技术，用于在免疫荧光研究中定量受损/死亡细胞。
