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Hoechst 33342 staining of multicell spheroids, three-dimensional cell clusters grown in vitro, results in a marked gradient of cellular fluorescent intensities inward from the spheroid periphery. The penetration of the dye is concentration and time dependent, so staining can be coupled with fluorescence activated cell sorting techniques to allow disaggregated single cells to be sorted or selected according to their degree of staining and therefore their depth within the spheroid. We have found the staining procedure to be highly reproducible, and to result in minimal toxicity even to the more brightly staining external cells. Comparison of this technique with others for cell selection suggests that increased resolution is available with the Hoechst technique.

Use of Hoechst 33342 for cell selection from multicell systems.