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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 micrograms/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 micrograms/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.

Original publication

DOI

10.1002/cyto.990150106

Type

Journal article

Journal

Cytometry

Publication Date

01/1994

Volume

15

Pages

28 - 34

Addresses

Department of Immunology Research, Merck Research Laboratories, Rahway, New Jersey 07065.

Keywords

B-Lymphocytes, Cells, Cultured, Animals, Mice, Inbred C57BL, Mice, Benzimidazoles, Ethidium, DNA, RNA, Intercalating Agents, Flow Cytometry, Ultraviolet Rays, Cell Cycle, Cell Division, Cell Survival, Female