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

Assessment of DNA content by flow cytometry has largely depended on st aining techniques which do not permit exclusion of dead cells from the data set. During studies of B cell activation in vitro, the large num ber of nonviable cells greatly affects the cell cycle distribution and thus the accurate evaluation of proliferation flow cytometry. This re port describes the development of two dual staining techniques which u se Hoechst 33342 and ethidium bromide excited by a single UV source to eliminate dead cells from the DNA histogram of the viable cells in mu rine B cell cultures. Hoechst 33342 and 0.62 mu g/ml of ethidium bromi de permit the evaluation of cell cycle distributions on the viable cel ls with a ratio gate. The combination of Hoechst 33342 and 6.2 mu g/ml ethidium bromide results in the resolution of the two populations due to fluorescence energy transfer with a single PMT. Using this techniq ue we demonstrated the simultaneous determination of DNA and RNA conte nt on viable cells using only two PMTs. Both these techniques can be p erformed on either a laser or an are lamp flow cytometer where CVs of less than 7% and as low as 3.2% are normally achieved. Determination o f the S phase using these techniques produces a high correlation with DNA synthesis determined by radiolabeled precursor determination. Thes e techniques permit the use of flow cytometry to determine proliferati on during B cell activation. (C) 1994 Wiley-Liss, Inc.