Isolation by fluorescence‐activated cell sorting of cells of a human lymphoblastoid cell strain containing mutations in the lambda immunoglobulin gene

Abstract

Fluorescence-activated cell sorting (FACS) was used to establish clonal populations of a human lymphoblastoid cell strain that contain spontaneously occurring and N-methyl-N'-nitro-N-nitrosoguanidine-induced mutations in the lambda immunoglobulin gene. Multiple rounds of FACS using a monoclonal antibody specific for the membrane-expressed human lambda immunoglobulin were used to enrich the population fraction of cells lacking a wild-type lambda immunoglobulin on the cell surface. Approximately 20% of the clonal populations established after five rounds of FACS-mediated enrichment did not express the lambda immunoglublin epitope recognized by the monoclonal antibody used for selection. However, evaluation of the FACS-selected mutant clonal populations with a polyclonal antilambda antibody, or a monoclonal antibody directed against a different epitope on the lambda immunoglobulin made by the T5-1 cells, indicated that the mutant clonal populations expressed lambda immunoglobulin on their cell surfaces. Additionally, the presence of lambda mRNA and of both secreted and cytoplasmic lambda protein confirmed the transcription and translation of the lambda immunoglobulin gene. These data suggest that FACS-mediated selection employing epitope-specific monoclonal antibodies provides a powerful technique for isolation of cell populations that express mutations within the coding region of the lambda immunoglobulin gene.

Novel electrophoretic protocol for collection of mutations in the lambda light chain immunoglobulin gene in a human B‐lymphoblastoid cell strain

“…The cell strain was routinely grown in RPMI 1640 medium (GIBCO, Grand Island, NY) supplemented with 15% defined fetal bovine serum (Hyclone, Logan, UT), 2 mM L-glutamine (GIBCO) and 48 pg/ml gentamicin (Elkins-Sinn, Cherry Hill, NJ). Stock cultures were maintained as described [7]. For characterization of secreted lambda light chain protein by isoelectric focusing (IEF), T5-1 cells were cultured in HBlOl medium (Hana Biologics, Berkeley, CA), a serum-free derivative of RPMI 1640 supplemented with 760 pg/ml albumin, 15 pdml transferrin, 25 pdml insulin, 2 mM glutamine, and 48 pg/ml gentamicin.…”

“…The proteins in the IEF gel were transferred overnight to a nitrocellulose membrane (BA 85, Schleicher and Schuell, Keene, NH). Lambda protein was identified using a commercial polyclonal anti-human lambda light chain antibody as the primary antibody [7]. The quantity of lambda immunoglobulin protein in cellular lysates on slot blots was determined by the same immunoassay used for western blots of IEF gels.…”

“…Integrity of lambda immunoglobulin mRNA was determined by northern blot analysis of formamide/formaldehyde gels and quantitation of lambda mRNA was determined by northern analysis of the average density of three independent vacuum slot blots using standard methods [7]. Lambda immunoglobulin mRNA was identified by probing blots with an 0.8 kb genomic fragment of a human lambda light chain immunoglobulin gene [9].…”

“…Flow cytometry and indirect immunofluorescence were used to determine the presence and relative quantity of lambda immunoglobulin and CD19 on cell membranes of the clonal populations [7]. The primary antibodies used were Leu-12, BDHL, and 2G3All.…”