Some of this material was presented at the XIII International Meeting of the Society for Analytical Cytology, Breckenridge, Colorado.
Single UV excitation of hoechst 33342 and ethidium bromide for simultaneous cell cycle analysis and viability determinations on in vitro cultures of murine B lymphocytes†
Article first published online: 8 MAR 2005
Copyright © 1994 Wiley-Liss, Inc.
Volume 15, Issue 1, pages 28–34, 1 January 1994
How to Cite
Boltz, R. C.“Dutch”., Fischer, P. A., Wicker, L. S. and Peterson, L. B. (1994), Single UV excitation of hoechst 33342 and ethidium bromide for simultaneous cell cycle analysis and viability determinations on in vitro cultures of murine B lymphocytes. Cytometry, 15: 28–34. doi: 10.1002/cyto.990150106
- Issue published online: 23 JUN 2005
- Article first published online: 8 MAR 2005
- Manuscript Accepted: 23 JUN 1993
- Manuscript Received: 5 AUG 1991
- Cell cycle analysis;
- FRCS analyzer;
- low viability cultures;
- B cell differentiation
Assessment of DNA content by flow cytometry has largely depended on staining techniques which do not permit exclusion of dead cells from the data set. During studies of B cell activation in vitro, the large number of nonviable cells greatly affects the cell cycle distribution and thus the accurate evaluation of proliferation flow cytometry. This report describes the development of two dual staining techniques which use Hoechst 33342 and ethidium bromide excited by a single UV source to eliminate dead cells from the DNA histogram of the viable cells in murine B cell cultures. Hoechst 33342 and 0. 62 μg/ml of ethidium bromide permit the evaluation of cell cycle distributions on the viable cells with a ratio gate. The combination of Hoechst 33342 and 6.2 μg/ml ethidium bromide results in the resolution of the two populations due to fluorescence energy transfer with a single PMT. Using this technique we demonstrated the simultaneous determination of DNA and RNA content on viable cells using only two PMTs. Both these techniques can be performed on either a laser or an arc lamp flow cytometer where CVs of less than 7% and as low as 3.2% are normally achieved. Determination of the S phase using these techniques produces a high correlation with DNA synthesis determined by radiolabeled precursor determination. These techniques permit the use of flow cytometry to determine proliferation during B cell activation. © 1994 Wiley-Liss, Inc.