Chapter 45 Cell Sorting with Hoechst or Carbocyanine Dyes as Perfmion Probes in Spheroids and Tumors

Publisher Summary The widespread availability of fluorescence-activated cell sorters (FACS) allows not only the quantitative demonstration of biological heterogeneity among the cells of tissues but also the ability to selectively recover cells with particular characteristics. Fluorescent probes that recognize variations in the expression of surface molecules, proteins, and DNA or RNA have become fairly commonplace; a number of functional stains have subsequently been developed that are responsive to membrane potential, respiratory activity, intracellular pH, and a growing list of other parameters. Recognition of the micro-environmental heterogeneities in growing spheroids prompted the development of techniques for the isolation of specific, defined cell subpopulations based on cellular position within the spheroid mass. Cell sorting techniques with Hoechst or carbocyanines as perfusion probes utilize the rapid binding of these agents as they diffuse from the medium into a spheroid or from the blood vessels into a tumor. Both agents remain localized in undamaged target cells after the multicell structure is disaggregated into a single-cell population. From this, it follows that sorting cells of given fluorescent intensities permits the recovery of subpopulations from the equivalently perfused regions of the spheroid or tumor system.