Simultaneous quantitation of hoechst 33342 and immunofluorescence on viable cells using a fluorescence activated cell sorter

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Abstract
The cytochemical stain Hoechst 33342 has been used to quantify DNA in viable cells and has been used under nonsaturating conditions to discriminate between lymphoid cell types. In order to correlate the quantitative emission from Hoechst 33342 with cell surface antigens, a fluorescence activated cell sorter was modified to simultaneusly detect emission from the UV excited Hoechst dy and fluorescein attached to the cell surface by immunofluorescence techniques. A special set of laser mmirrors was installed in an argon ion laser so that all the lines from 351‐488 nm could be used to illuminate the cells. Appropriate emission filters were used to separate the light emitted by Hoechst 33342 from the fluorescein. An electronic cross‐over circuit was used to compensate for special overlap between the two dyes. Analysis of murine lymph node cells stained both with Hoechst 33342 under nonsaturating conditions and anti‐Thy 1.2 indicated that the cells that stained dimly with the Hoechst dy expressed the Thy 1.2 marker while the cells that were brightly stained with Hoechst 33342 lacked this differentiation antigen. The correlation of cell surface myeloma protein with cell cycle on an in vitro cell line indicated that the heterogeneity of cell surface antigen expression could not be accounted for solely by variations occurring during the cell cycle.