Preparation of intact, full-size RNA from tissues or cells requires stringent precautions against ubiquitous and rather stable RNases. Fluorescence-activated cell sorting (FACS) usually aims at the isolation of cells according to cell surface markers on living cells, from which RNA can be obtained by standard protocols. The separation of cells according to intracellular immunofluorescence markers, such as intranuclear, intracytoplasmic, or secreted molecules, requires permeation of the cell membrane for the staining antibodies, which is usually achieved by fixation. However, commonly used fmatives such as ethanol, methanol, or formaldehyde do not inactivate RNases completely, thereby hampering the analysis of complete RNA molecules from fixed cells. We report isolation of intact, fullsize RNA suitable for Northern blotting from cells that were fixed by 95% ethanoY5% acetic acid containing RNase inhibitors, stained intracellularly, and sorted by FACS. o 1995 wiley-Liss, Inc.

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We have modified a FACS I by addition of a tibodies labelled with FITC or Texas Red, we tunable dye laser and an optical system for have isolated from a murine T-cell lymphoma fluorescence detection that allows physically line variant subclones expressing structurally independent measurement of green and red altered histocompatibility class I (H-2Kk) immunofluorescence. These modifications are molecules. inexpensive and should be applicable to most single-laser systems. By using the modified machine and double-fluorescence activated Key words: Dual immunofluorescence, cell selection with H-2Kk-specific monoclonal an-sorting, Texas Red, H-2 variantsThe experiments reported in this paper were aimed at isolating structural variants from the mouse lymphoma line LDHB that express altered instead of wild type H2Kk molecules on the cell surface. These variants are interesting for the investigation of the structural basis of class I antigen function in immune recognition. Furthermore, they can be used to approach the question of which types of somatic mutation occur in the polymorphic family of class I genes.In our previous work, we had achieved this aim by inhibiting the binding of one fluorescent monoclonal anti-Kk antibody to the cells by an excess of a second, unlabelled one and selecting for rare fluorescent cells in the fluorescence activated cell sorter (FACS) (10). This approach was restricted to the selection of structural variants that had lost the expression of one of two Kk determinants, which had to be sufficiently close to each other (topographically) so that the binding of an antibody to one of them would inhibit the binding of a second antibody to the other. In the present work this limitation was overcome by staining wild type cells with two monoclonal anti-Kk antibodies that are labelled with green and red fluorescent tags, respectively, and enriching the cells that carry one label only. For this purpose, we have upgraded a single-laser FACS I instrument by addition of a dye laser that supplies the excitation light for red-fluorescent dyes, such as Texas Red or propidium iodide, that can be used for dual parameter sorting with fluorescein as a green fluorescent label. In contrast to other approaches to double (or triple) fluorescence (161, the dye laser is pumped by the same argon laser that provides the 488 nm light for fluorescein excitation. MATERIALS AND METHODS Cell SorterThe modifications described here were added to a FACS I instrument (Becton Dickinson, Mountain View, CA; serial no. 04). A 5-watt argon ion laser (SpectraPhysics Model 164-08, Darmstadt, FRG) operated at 4 Watts in an all-line mode to give approximately 800 milliwatts at 488 nm and 1.7 watt at 514 nm served as a source for 488 nm excitation of fluorescein and for pumping a Model 375 dye laser (Spectra-Physics, Darmstadt, FRG). The latter was operated with Rhodamin 6G Cambda Physik, Gottingen, FRG) in a setup as described by . Reflectors, dichroic mirrors, and interference and coloured-glass filters used in the system (Fig. 1)

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