IMPROVED SENSITIVITY IN FLOW CYTOMETRIC INTRACELLULAR IONIZED CALCIUM MEASUREMENT USING FLUO-3 FURA RED FLUORESCENCE RATIOS

被引:107
作者
NOVAK, EJ [1 ]
RABINOVITCH, PS [1 ]
机构
[1] UNIV WASHINGTON,DEPT PATHOL,SM-30,SEATTLE,WA 98195
来源
CYTOMETRY | 1994年 / 17卷 / 02期
关键词
KINETIC ANALYSIS; RATIOMETRIC CALCIUM ANALYSIS; INDO-1; FLOW CYTOMETRY;
D O I
10.1002/cyto.990170205
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:135 / 141
页数:7
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