Measurement of DNA and antigen expression of live cells using three fluorochromes and two detectors

Abstract

In a multiparameter flow cytometric study, the fluorescence from the two different dyes fluorescein and dansyl-lysine were detected in the same spectral interval on the same photomultiplier tube, using two lasers to excite the fluorochromes at two separate laser foci. The two signals were split by a T-cable at the output of the photomultiplier tube and synchronized by delaying the first signal. Dansyl-lysine binds to the membrane of heat-inactivated cells and was used to distinguish between live and dead cells after a hyperthermic treatment. By gating on the dansyl-lysine signal, the DNA distribution and surface antigenexpression of live and dead cells were obtained separately, using Hoechst 33342 and a monoclonal antibody conjugated to fluorescein. Key terms: Dansyl-lysine, Hoechst 33342, immunofluorescenceCellular uptake of various dyes is a rapid and oftenused method to estimate cell viability. Rice et al. (6) compared the uptake of several dyes after hyperthermia, and compared the percentage of negatively stained cells with cell survival measured by colony formation. All the dyes studied underestimated heat-induced cell killing as measured by colony formation. The fluorochrome dansyl-lysine was found to be the best indicator for cell killing after hyperthermia; it is considerably better than, for instance, propidium iodide, especially after heating with doses that kill more than 90% of the cells (6). Several agents, which primarily affect the plasma membrane, are reported to induce dansyl-lysine staining, while agents killing cells through other mechanisms do not induce such staining (7).By flow cytometry, dansyl-lysine negative and positive cells can be separated, thus distinguishing between live and dead cells. The objective of the present work was to develop a method to identify and characterize subpopulations of cells sensitive or resistant to hyperthermia. The method combines dansyl-lysine with Hoechst 33342 and monoclonal antibodies conjugated to fluorescein isothiocyanate (FITC), to measure DNA content and antigen expression of the live and dead cell populations. MATERIALS AND METHODS CellsThe murine B-cell lymphoma 38C13, a carcinogen [dimethyl-benz(a)anthracene]-induced cell line (21, was used, since the present work was part of a study of heat-induced changes of the expression of idiotype antigen on B-cells (3). The cell line was provided by Dr. R. Levy (Stanford University). Cells were grown in suspension in RPMI 1640 medium (Gibco, Grand Island, NY) supplemented with 10% fetal calf serum (FCS) (Irvine Scientific, Santa Ana, CAI, penicillin-streptomycin, L-glutamine, and 5 x lop5 M 2-mercaptoethanol (Sigma, St. Louis, MO). The cultures were kept a t 37°C in an atmosphere of 5% COz. Heat TreatmentCell suspensions (5 x lo5 cellsiml) were heated at 43°C for 30 min in complete medium in a thermostatically regulated water bath. The cells were flushed with 5% CO, before heating. The pH during heating was 7.2 t 0.1. StainingThe 38C13 cell line expresses IgM with a n unique idiotype. The monoclonal anti...