Flow cytometric measurement of fluorescence (Forster) resonance energy transfer from cyan fluorescent protein to yellow fluorescent protein using single-laser excitation at 458 nm
Flow cytometric measurement of fluorescence (Forster) resonance energy transfer from cyan fluorescent protein to yellow fluorescent protein using single-laser excitation at 458 nm
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DOI：
10.1002/cyto.a.10037
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发表时间：
2003-05-01
期刊：
影响因子：
3.7
通讯作者：
Lipsky, PE
中科院分区：
文献类型：
--
作者：
He, LS;Bradrick, TD;Lipsky, PE
背景资料：使用不同的绿色荧光蛋白（GFP）变体允许通过荧光（Forster）共振能量转移（FRET）研究活转染细胞中的蛋白质-蛋白质相互作用和共定位。流式细胞术是检测FRET的敏感方法。然而，在流式细胞FRET测定中使用的典型的双激光方法通常不适用，因为它们需要专门的氪紫外（UV）激光。这项工作的目的是开发一种流式细胞术的方法来检测荧光共振能量转移之间的青色荧光蛋白（CFP;供体）和黄色荧光蛋白（YFP;受体），通过使用458 nm的激发从一个单一的可调氩离子laser.Methods：融合蛋白结合蛋白（FBP）相互作用阻遏物（FIR）和FBP是c-myc转录因子，是已知的物理相互作用。为了研究它们在活细胞内的相互作用，FIR和FBP的结合基序，FBP中央结构域（FBPcd），分别与CFP和YFP融合，这对荧光标记的蛋白质用于检测FRET在体内。用编码CFP-FIR融合蛋白和YFP的表达质粒转染的细胞作为阴性对照，CFP-YFP融合蛋白作为阳性对照，或CFP-FIR和YFP-FBPcd融合蛋白，在用来自可调谐氩离子激光器的458 nm线激发后，检查FRET。FRET测量为YFP：CFP发射的比率或在564606 nm处的YFP发射。常规FRET使用413 nm紫外线从氪激光进行了检查比较。荧光信号用定制的光学滤波器配置分离，除了488/30 nm（CFP）、530/30 nm（YFP）和585/42 nm（FRET）带通滤波器之外，还使用530 nm短通、500 nm长通和560 nm短通二色性。此外，激光扫描共聚焦显微镜光漂白技术被用来记录FRET发生显示，供体CFP荧光强度增加后，其受体YFP被光漂白。结果：在458 nm氩离子激光激发下，转染CFP-YFP或CFP-FIR和YFP-FBPcd的细胞中，受体YFP信号增强，CFP信号减弱。类似地，当在564 - 606 nm处评估YFP发射时，在这些条件下检测到FRET。在YFP：CFP比率的增加和在564 - 606 nm处检测到的YFP发射之间观察到强相关性，这与通过两种方法同时检测到FRET的结论一致。还使用常规的流式细胞术氪紫外激光技术来确认CFP-YFP融合蛋白和CFP-FIR YFP-FBPcd的FRET发生。FRET也证实了共聚焦光漂白技术，其中供体CFP强度增强后，其受体YFP被光漂白。流式细胞仪和共聚焦显微镜的结果证实了由spectrofluorometry.Conclusion：这些结果表明，荧光共振能量转移信号的流式细胞仪检测的可行性，从CFP到YFP的激发与458 nm线从可调谐氩离子激光。该方法是有效的激发与氪紫外激光，因此应该使FRET更普遍可用的流式细胞术技术。细胞计数A部分53 A：39-54，2003。发布于2003年Wiley-Liss，Inc.（匕首）
Background: Use of distinct green fluorescent protein (GFP) variants permits the study of protein-protein interactions and colocalization in viable transfected cells by fluorescence (Forster) resonance energy transfer (FRET). Flow cytometry is a sensitive method to detect FRET. However, the typical dual-laser methods used in flow cytometric FRET assays are not generally applicable because they require a specialized krypton ultraviolet (UV) laser. The purpose of this work was to develop a flow cytometric method to detect FRET between cyan fluorescent protein (CFP; donor) and yellow fluorescent protein (YFP; acceptor) by using the 458-nm excitation from a single tunable argon-ion laser.Methods: FUSE-binding protein (FBP) interacting repressor (FIR) and FBP are c-myc transcription factors and are known to interact physically. To examine their interaction within viable cells, FIR and the binding motif of FBP, the FBP central domain (FBPcd), were fused with CFP and YFP, respectively, and this pair of fluorescently-tagged proteins was used to detect FRET in vivo. Cells transfected with expression plasmids encoding a CFP-FIR fusion protein and YFP as a negative control, a CFP-YFP fusion protein as a positive control, or CFP-FIR and YFP-FBPcd fusion proteins were examined for FRET after excitation with a 458-nm line from a tunable argon-ion laser. FRET was measured as the ratio of YFP:CFP emission or as YFP emission at 564606 nm. Conventional FRET using the 413-nm UV line from a krypton laser was examined for comparison. Fluorescence signals were separated with a customized optical filter configuration using 530-nm shortpass, 500-nm longpass, and 560-nm shortpass dichroics in addition to 488/30 nm (CFP), 530/30 nm (YFP), and 585/42 nm (FRET) bandpass filters. Further, a laser-scanning confocal microscopic photobleach technique was used to document that FRET occurred by showing that the intensity of donor CFP fluorescence increased after its acceptor YFP was photobleached. Steady-state spectrofluorometry was used to confirm and validate the results detected by flow cytometry.Results: Upon excitation with the 458-nm line of the argon-ion laser, the enhancement of the acceptor YFP signal and the decrease of the CFP signal were easily detected in cells transfected with the CFP-YFP construct or CFP-FIR and YFP-FBPcd. Similarly, FRET was detected under these conditions when the YFP emission was assessed at 564 - 606 nm. A strong correlation was observed between the increase in the YFP:CFP ratio and the YFP emission detected at 564 - 606 nm, consistent with the conclusion that FRET was detected comparably by both methods. A conventional flow cytometric krypton UV-laser technique was also used to confirm that FRET occurred with the CFP-YFP fusion protein and from CFP-FIR YFP-FBPcd. FRET also was confirmed by a confocal photobleaching technique, in which donor CFP intensity was enhanced after its acceptor YFP was photobleached. The flow cytometric and confocal microscopic results were confirmed by spectrofluorometry.Conclusion: These results demonstrated the feasibility of flow cytometric detection of FRET signals from CFP to YFP by excitation with the 458-nm line from the tunable argon-ion laser. The method was as efficient as excitation with the krypton UV laser and therefore should make FRET a more generally available flow cytometric technique. Cytometry Part A 53A:39-54, 2003. Published 2003 Wiley-Liss, Inc.(dagger)