IMPROVED SENSITIVITY IN FLOW CYTOMETRIC INTRACELLULAR IONIZED CALCIUM MEASUREMENT USING FLUO-3 FURA RED FLUORESCENCE RATIOS
IMPROVED SENSITIVITY IN FLOW CYTOMETRIC INTRACELLULAR IONIZED CALCIUM MEASUREMENT USING FLUO-3 FURA RED FLUORESCENCE RATIOS
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DOI：
10.1002/cyto.990170205
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发表时间：
1994-10-01
期刊：
影响因子：
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通讯作者：
RABINOVITCH, PS
中科院分区：
文献类型：
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作者：
NOVAK, EJ;RABINOVITCH, PS
测量细胞内离子钙浓度（[Ca2+](i)）的变化已被证明在研究细胞对激活刺激的反应中具有广泛的应用。荧光染料Indo-1已经成功地用于流式细胞术，当用作比例指示剂时，它提供了最佳的灵敏度和准确性。不幸的是，这种染料需要紫外线（UV）激发，而这通常是不可用的。我们在这里表明，通过同时加载Fura red和fluo-3染料，可以获得类似的结果。Fura Red和fluo-3都是使用常见的蓝色488 nm激光线激发的。在两种染料浓度适当的情况下，fluo-3/Fura Red比的响应幅度大于indo-1，而测量的细胞间变化与indo-1相似。分析可以与pe标记抗体的免疫荧光检测同时结合，以实现细胞亚群内的[Ca2+](i)测量。(C) 1994 Wiley-Liss, Inc。
Measurement of changes in intracellular ionized calcium concentrations ([Ca2+](i)) has proved to be of wide use in the study of cellular responses to activating stimuli. The fluorescent dye Indo-1 has successfully been used in flow cytometry for this purpose, and when used as a ratiometric indicator it provides optimum sensitivity and accuracy. Unfortunately, this dye requires ultraviolet (UV) excitation which is often not available. We show here that similar results can be obtained using a ratio of green to red fluorescence from the simultaneous loading of the dyes Fura Red and fluo-3. Both Fura Red and fluo-3 are excited using the commonly available blue 488 nm laser Line. With appropriate concentrations of the two dyes, the magnitude of response with the fluo-3/Fura Red ratio is greater than that achieved with indo-1, while the intercellular variation in measurement is similar to that seen with indo-1. Analyses can be simultaneously combined with immunofluorescent detection of PE-labeled antibodies to enable [Ca2+](i) measurement within cell subsets. (C) 1994 Wiley-Liss, Inc.