Analysis of subcellular sized particles - Capillary electrophoresis with post-column laser-induced fluorescence detection versus flow cytometry

被引:16
作者
Poe, Bobby G. [1 ]
Navratil, Marian [1 ]
Arriaga, Edgar A. [1 ]
机构
[1] Univ Minnesota, Dept Chem, Minneapolis, MN 55455 USA
关键词
flow cytometry; capillary electrophoresis; laser-induced fluorescence; signal-to-noise ratio; mitochondria;
D O I
10.1016/j.chroma.2006.10.011
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Flow cytometry (FCM) and more recently capillary electrophoresis with post-column laser-induced fluorescence detection (CE-LIF) have both been used for subcellular particle analysis but their analytical performance has not been compared. In this work, we compare a commercial FCM with an in-house built CE-LIF instrument using fluorescently labeled microspheres and isolated mitochondria. As evidenced by the relative standard deviation (RSD) of the individual fluorescence intensities, FCM is two-fold better than CE-LIF for microspheres with >= 1.5 x 106 molecules of equivalent soluble fluorescein (MESF). However, FCM has a comparatively low signal-to-noise ratio (S/N) and high RSD for microspheres with < 1.5 x 10(6) MESE CE-LEF, on the other hand, produces S/N ratios that are > 25 times higher than FCM for all the microspheres tested and a lower RSD for microspheres with < 1.5 x 10(6) MESR When 10-N-nonyl acridine orange (NAO)-labeled mitochondria are analyzed, the S/N ratios of both techniques are similar. This appears to result from photobleaching of NAO-labeled mitochondria as they are detected by the LIF detector of the CE-LIF instrument. Both techniques have a niche in subcellular analysis; FCM has the advantage of collecting data for thousands of particles quickly, whereas CE-LIF consumes less than a nanoliter of sample and provides the electrophoretic mobility for individual particles. (c) 2006 Elsevier B.V. All rights reserved.
引用
收藏
页码:249 / 255
页数:7
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