Inexpensive upgrading of a FACS I and isolation of rare somatic variants by double-fluorescence sorting.

We have modified a FACS I by addition of a tunable dye laser and an optical system for fluorescence detection that allows physically independent measurement of green and red immunofluorescence. These modifications are inexpensive and should be applicable to most single-laser systems. By using the modified machine and double-fluorescence activated selection with H-2Kk-specific monoclonal antibodies labelled with FITC or Texas Red, we have isolated from a murine T-cell lymphoma line variant subclones expressing structurally altered histocompatibility class I (H-2Kk) molecules.

Analysis by flow cytometry of DNA synthesis during the life cycle of African trypanosomes.

DNA content, at different stages in the life cycle of the hemoprotozoan parasite Trypanosoma brucei, has been analysed with a fluorescence activated cell sorter. It was observed that the long slender bloodstream form stage and procyclic culture forms (analogous to the tsetse fly midgut stage) are dividing cell populations with cells in G1, S, G2 and mitosis. Short stumpy bloodstream form and metacyclic fly salivary gland form populations are composed of non-dividing parasites stabilized in G1 or G0 of the cell cycle. Haploids, possible sexual forms, were not detected. In response to transfer to a culture system which mimics the fly midgut, short stumpy bloodstream form parasites were readily able to initiate DNA synthesis and differentiate into dividing procyclic culture forms. This supports the suggested role of the short stumpy form as a transitional stage between the mammalian host and the tsetse fly vector. Analysis of early and late bloodstream populations of another salivarian trypanosome, Trypanosoma vivax, revealed a transition from dividing to stationary cell population similar to that observed with T. brucei. A hitherto unrecognized morphological form of T. vivax, analogous to the T. brucei short stumpy form, was detected. It is suggested that the long slender to short stumpy morphological transformation, long known in T. brucei, reflects a physiological transition from dividing to nondividing parasite relevant to the life cycle of all the salivarian trypanosomes.

Switch from NP-specific IgG3 to IgG1 in the mouse hybridoma cell line S24/63/63.

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.

Isolation of variants of mouse myeloma X63 that express changed immunoglobulin class.

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, kappa). In the wild-type cell population, only one type of variants was found, namely, cells expressing IgG2b. From an IgG2b variant clone we isolated secondary variants that had either reverted to IgGI expression or expressed IgG2a or IgG2a and IgG2b concomitantly. The variant heavy chains are of normal size. The variant immunoglobulins were characterized serologically, and all of them still expressed the wild-type idiotype. Wild-type and variant cell populations were screened for heavy chain class-switch variants by fluorescence microscopy. A variety of switch variants was found in addition to the ones isolated by cell sorting, and a clear pattern of class switching (gamma 1 leads to gamma 2b leads to gamma 2a leads to alpha) with frequent reversion emerges from this analysis. Cells expressing new heavy chain classes occurred at frequencies of about 10(-7)-10(-6)/cell per generation, whereas revertants were as frequent as 10(-6)-10(-5)/cell per generation.

A new mouse myeloma cell line that has lost immunoglobulin expression but permits the construction of antibody-secreting hybrid cell lines.

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.

Isolation of myeloma variants with predefined variant surface immunoglobulin by cell sorting.

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.