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

Bobby G. Poe, Marian Navratil, Edgar A. Arriaga

Research output: Contribution to journalArticlepeer-review

18 Scopus citations

Abstract

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 × 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 × 106 MESF. CE-LIF, 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 × 106 MESF. 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.

Original languageEnglish (US)
Pages (from-to)249-255
Number of pages7
JournalJournal of Chromatography A
Volume1137
Issue number2
DOIs
StatePublished - Dec 29 2006

Bibliographical note

Funding Information:
This work was supported by the National Institute of Health (R01-AG20866). B.P. acknowledges support from NIH-Chemistry/Biology Interface Training Grant (GM08700). E.A. is supported by an NIH Career Award (1K02-AG21453). The authors thank the Flow Cytometry Core Facility of the University of Minnesota Cancer Center, supported by the National Cancer Institute (P30 CA77598).

Keywords

  • Capillary electrophoresis
  • Flow cytometry
  • Laser-induced fluorescence
  • Mitochondria
  • Signal-to-noise ratio

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