We have used fluorescence-activated cell sorting with class-specific antisera to isolate spontaneous variants in the expression of immunoglobulin heavy chain class from the mouse myeloma cell line X63 (IgGi, K pattern of class switching (y -_ y2b -_ 'y2a -_ a) with frequent reversion emerges from this analysis. Cells ex ressing new heavy chain classes occurred at frequencies ofabout 10-7-10-6/cell per generation, whereas revertants were as frequent as 10-6-10-5/cell per generation.

We describe a procedure for the isolation of somatic cell variants in which gene products are expressed on the cell surface that are not expressed in the wild type. Cloned cells of the myeloma line MPC 11, which expresses an IgG2b protein, were incubated with an antiserum specific for IgGI and IgG2a. Cells reacting with this antiserum were stained with a fluorescent anti-antiserum and enriched in three cycles of sorting in the fluorescence-activated cell sorter and subsequent growth in vitro. From the enriched population two variants were isolated by cloning in soft agar. One of them expressed a variant immunoglobulin that types serologically as an IgG2a but whose variable portion was idiotypically related to that of the MPC 11 wild-type protein.

We have isolated a subclone of the mouse myeloma cell line P3-X63-Ag8 that does not express immunoglobulin heavy or light chains. This clone X63-Ag8.653 can be used for efficient fusion with antibody-forming cells to obtain hybrid cell lines producing pure monoclonal antibodies. Screening of hybrid cell lines for specificity and immunoglobulin classes was done with a modified enzyme-linked immunosorbent assay.

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)

We have isolated by fluorescence-activated cell sorting an IgG1 expressing class switch variant (S24-1/47) from the IgG3-producing mouse hybridoma cell line S24/63/63. Both Ig bind the hapten 4-hydroxy-3-nitro-phenylacetyl (NP) with approximately the same affinity and fine specificity and are idiotypically indistinguishable. The apparent m.w. is 50,000 for the gamma 1 and 51,000 for the gamma 3 chain. The genomic DNA of IgG3-producing S24/63/63 cells contains a 14-kb Eco RI restriction fragment with C gamma 3 sequences representing a rearranged C gamma 3 gene. In the class switch variant S24-1/47 the C gamma 3 gene is deleted.