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Simultaneous analysis of relative DNA and glutathione content in viable cells by phase-resolved flow cytometry

1999; Wiley; Volume: 35; Issue: 1 Linguagem: Inglês

10.1002/(sici)1097-0320(19990101)35

1097-0320

Jan F. Keij, Carolyn Bell-Prince, John A. Steinkamp,

Photoreceptor and optogenetics research

CytometryVolume 35, Issue 1 p. 48-54 Original ArticleFree Access Simultaneous analysis of relative DNA and glutathione content in viable cells by phase-resolved flow cytometry Jan F. Keij, Corresponding Author Jan F. Keij [email protected] Life Sciences Division, Los Alamos Laboratory, Los Alamos, New MexicoLos Alamos National Laboratory, LS-5, MS M888, Los Alamos, NM 87545.Search for more papers by this authorCarolyn Bell-Prince, Carolyn Bell-Prince Life Sciences Division, Los Alamos Laboratory, Los Alamos, New MexicoSearch for more papers by this authorJohn A. Steinkamp, John A. Steinkamp Life Sciences Division, Los Alamos Laboratory, Los Alamos, New MexicoSearch for more papers by this author Jan F. Keij, Corresponding Author Jan F. Keij [email protected] Life Sciences Division, Los Alamos Laboratory, Los Alamos, New MexicoLos Alamos National Laboratory, LS-5, MS M888, Los Alamos, NM 87545.Search for more papers by this authorCarolyn Bell-Prince, Carolyn Bell-Prince Life Sciences Division, Los Alamos Laboratory, Los Alamos, New MexicoSearch for more papers by this authorJohn A. Steinkamp, John A. Steinkamp Life Sciences Division, Los Alamos Laboratory, Los Alamos, New MexicoSearch for more papers by this author First published: 06 January 1999 https://doi.org/10.1002/(SICI)1097-0320(19990101)35:1 3.0.CO;2-5Citations: 15AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat Abstract Background: Analysis of the DNA cell cycle and glutathione content cannot be performed on viable cells, because the fluorescence emissions of the DNA-specific probe Hoechst 33342 and the glutathione-specific probe monobromobimane overlap completely. We decided to explore whether the emissions could be resolved by the singlet excited state lifetimes of the probes. Methods: Viable cells were first incubated with Hoechst 33342 at 37°C for 30 min and then with monobromobimane at room temperature for 10 min. Samples were excited with a sinusoidally modulated laser beam (10 MHz) in a flow cytometer. The Hoechst 33342 and monobromobimane lifetimes and fluorescence intensities were resolved by using phase-sensitive detectors. Results: The observed singlet excited state lifetimes were 1.5 ns for Hoechst 33342 and 12 ns for monobromobimane. The glutathione (GSH) content was shown to increase as cells (GM130, HL60, U937) progressed through the cell cycle. However, after the data were corrected for differences in cell volume, it was found that the GSH concentration was constant throughout the cell cycle of the exponentially growing cells. Conclusions: Phase-resolved flow cytometry provides a means for the specific analysis of the GSH content/concentration as a function of the cell's position in the DNA cell cycle in viable cells. Cytometry 35:48–54, 1999. © 1999 Wiley-Liss, Inc. LITERATURE CITED 1 Durand RE, Olive PL. Flow cytometric techniques for studying cellular thiols. Radiat Res 1983; 95: 456–470. Medline 2 O'Connor JE, Kimler BF, Morgan MC, Tempas KJ. A flow cytometric assay for intracellular nonprotein thiols using mercury orange. Cytometry 1988; 529–532.–. 3 Poot M, Kavanagh J, Kang HC, Haugland RP, Rabinovitch PS. 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