Hawley T S, Telford W G, Ramezani A, Hawley R G
Jerome H. Holland Laboratory for the Biomedical Sciences, American Red Cross, Rockville, MD, USA.
Biotechniques. 2001 May;30(5):1028-34. doi: 10.2144/01305rr01.
Flow cytometric procedures are described to detect a "humanized" version of a new red fluorescent protein (DsRed) from the coral Discosoma sp. in conjunction with various combinations of three Aequorea victoria green fluorescent protein (GFP) variants--EYFP, EGFP, and ECFP. In spite of overlapping emission spectra, the combination of DsRed with EYFP, EGFP, and ECFP generated fluorescence signals that could be electronically compensated in real time using dual-laser excitation at 458 and 568 nm. Resolution of fluorescence signals from DsRed, EYFP, and EGFP was also readily achieved by single-laser excitation at 488 nm. Since many flow cytometers are equipped with an argon-ion laser that can be tuned to 488 nm, the DsRed/EYFP/EGFP combination is expected to have broad utility for facile monitoring of gene transfer and expression in mammalian cells. The dual-laser technique is applicable for use on flow cytometers equipped with tunable multiline argon-ion and krypton-ion lasers, providing the framework for studies requiring simultaneous analysis of four fluorescent gene products within living cells.
描述了流式细胞术程序，用于检测来自珊瑚盘碟藻属（Discosoma sp.）的一种新型红色荧光蛋白（DsRed）的“人源化”版本，以及三种维多利亚多管水母绿色荧光蛋白（GFP）变体——增强型黄色荧光蛋白（EYFP）、增强型绿色荧光蛋白（EGFP）和增强型青色荧光蛋白（ECFP）的各种组合。尽管发射光谱存在重叠，但DsRed与EYFP、EGFP和ECFP的组合产生了荧光信号，这些信号可以通过在458和568 nm处的双激光激发进行实时电子补偿。通过在488 nm处的单激光激发也很容易实现对DsRed、EYFP和EGFP荧光信号的分辨。由于许多流式细胞仪配备了可调节至488 nm的氩离子激光器，因此DsRed/EYFP/EGFP组合有望广泛用于便捷监测哺乳动物细胞中的基因转移和表达。双激光技术适用于配备可调谐多线氩离子和氪离子激光器的流式细胞仪，为需要在活细胞内同时分析四种荧光基因产物的研究提供了框架。
