Multiparameter analysis of human epithelial tumor cell lines by laser scanning cytometry

Laser scanning cytometry (LSC) is a relatively new slide-based technology d eveloped for commercial use by CompuCyte (Cambridge, MA) for performing mul tiple fluorescence measurements on individual cells. Because techniques dev eloped for performing four or more measurements on individual lymphoid cell s based on light scatter as a triggering parameter far cell identification are not suitable for the identification of fixed epithelial tumor cells, an alternative approach is required for the analysis of such cells by LSC. Me thods for sample preparation, event triggering, and the performance of mult iple LSC measurements on disaggregated fixed human cells were developed usi ng normal lymphocytes and two human breast cancer cell lines, JC-1939 and M CF-7, as test populations. Optimal conditions for individual cell identific ation by LSC were found to depend on several factors, including deposited c ell density (cells per unit area), the dynamic range of probe fluorescence intensities, and intracellular distribution of the fluorescent probe. Spars ely deposited cells exhibited the least cell overlap and the brightest immu nofluorescent staining. Major advantages of using DNA probes over a cytopla smic immunofluorescent protein marker such as tubulin for event triggering are that the former exhibit greater fluorescence intensity within a relativ ely sharply demarcated nuclear region. The DNA-binding dye LDS-751 was foun d to he suboptimal for quantitative DNA measurements but useful as a trigge ring measurement that permits the performance of simultaneous fluorescein i sothiocyanate-, phycoerythrin-, and indodicarbocyanine-based measurements o n each cell. A major potential advantage of LSC over flow cytometry is the high yields of analyzable cells by LSC, permitting the performance of multi ple panels of multicolor measurements on each tumor. In conclusion, we have developed and optimized a technique for performing multiple fluorescence m easurements on fixed epithelial cells by LSC based on event triggering usin g the DNA-binding dye LDS 751. Although not ideal for quantitative measurem ents of cell DNA content, the large Stokes shift of this dye permits the pe rformance of three or more additional fluorescence measurements on each cel l. Cytometry (Comm. Clin. Cytometry) 42: 347-356, 2000. (C) 2000 Wiley-Liss , Inc.