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

Abstract

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.

Fluorescence characteristics of 5-carboxytetramethylrhodamine linked covalently to the 5' end of oligonucleotides: multiple conformers of single-stranded and double-stranded dye-DNA complexes

“…Much effort has been spent in determining the affinity, specificity, and structures of these DNA-dye complexes. Because the fluorescence properties of these dye molecules are often dramatically affected by their interactions with nucleic acids, the quantum yields and the polarization of fluorescence can be utilized to detect and quantify nucleic acids rapidly with samples at low concentration or with microscale quantities, as in flow cytology and fluorescence microscopy (Arndt-Jovin and Jovin, 1990; Boltz et al, 1994;Hirons et al, 1994). In addition, many studies have been carried out using these DNA-binding dyes to determine physical characteristics of the nucleic acids themselves (Bresloff and Crothers, 1975; Ryan and Crothers, 1984;Macgregor et al, 1985Macgregor et al, , 1987Schurr et al, 1992).…”

Flow cytometric analysis of live cell proliferation and phenotype in populations with low viability

“…Two methods for measurement of cell-cycle distributions by DNA flow cytometry combined with dead cell exclusion have been described previously (4,37), but each has a disadvantage. The technique described by Muirhead et al requires preincubation of the cells before staining with deoxyribonuclease to permit discrimination of dead cells from live cells by decreased DNA content (4); however, this step adds approximately 90 min to the sample preparation time.…”

“…The technique described by Muirhead et al requires preincubation of the cells before staining with deoxyribonuclease to permit discrimination of dead cells from live cells by decreased DNA content (4); however, this step adds approximately 90 min to the sample preparation time. Another method uses combined staining of ethidium bromide and Hoechst 33342 for measurement of DNA content on viable cells (37); for this assay, both dyes, however, require ultraviolet excitation, which is not available on most benchtop flow cytometers.…”

Improving the resolution of cryopreserved X- and Y-sperm during DNA flow cytometric analysis with the addition of Percoll to quench the fluorescence of dead sperm.

“…A method to exclude dead cells from a sorting window based on the principle of dead cell gating was first reported by Boltz et al (1994) for lymphocytes; in these experiments the Hoechst fluorescence of the dead cells was quenched by ethidium bromide or PI. The technique has also been applied to mammalian sperm .…”