Fluorescence polarization of six membrane probes in embryonal carcinoma cells after differentiation as measured on a FACS II cell sorter

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Abstract
Fluorescence polarization measurements on a FACS II cell sorter were compared with static measurements on a spectrofluorimeter using calibration solutions and Hoechst 33258-labeled cells. For the flow cytometric measurements on the FACS we used a pseudodepolarizer for normalization of the output of the two photomultipliers. The results showed that fluorescein and fluoresceinated bovine serum albumin (BSA) solutions gave identical values on both instruments. The mean value for fluorescence polarization of Hoechst 33258-labeled cells as measured on the FACS was the same as the value obtained with the spectrofluorimeter. Subsequently the fluorescence polarization of six different membrane probes was determined using differentiating embryonal carcinoma cells as a model system. Differentiation was induced by treatment of the cells with retinoic acid together with cyclic AMP. With diphenylhexatriene (DPH) the fluorescence polarization increased from I‖/I = 1.55 to 1.74 upon differentiation. With a charged analog of DPH (TMA-DPH) fluorescence polarization increased from I‖/I = 1.87 to 2.02. No appreciable changes in fluorescence polarization were observed in this cell system when anthroyloxysterate probes (12-AS, 9-AS, 6-AS, 2-AS) were used.