Cloning of mouse myeloma cells and detection of rare variants

Abstract

Cultured mouse myeloma cells have been cloned in soft agar using a modification of the method established by Pluznik and Sachs ('65, '66) and by Bradley and Metcalf ('66). A linear relationship existed between the number of cells plated and the number of colonies produced. Conditions for obtaining optimum cloning efficiency and colony size were determined for the MPC-11 cell line. Feeder cells of mouse, human and rabbit origin and conditioned growth medium obtained from mouse cultures all had a n enhancing effect on colony formation. Immunoglobulin production by cloncd cells was detected by overlaying the clones with anti-immunoglobulin antiserum. The antiserum had no adverse effect on cloning efficiency or colony size. A reconstruction experiment was performed to show that the plate assay could reliably detect rare variants of immunoglobulin producing cells. The plate assay was validated by studying immunoglobulin production following recovery of clones from dishes and their growth to mass suspension culture. Immunoglobulin formation i n these cultures was assessed by a Ouchterlony immunodiffusion of the supernatant medium. and bv incubating the cells with radioactive amino acids and analyzing the intracellular acrylamide gels.

Monocyte receptors for fibronectin characterized by a monoclonal antibody that interferes with receptor activity.

“…Hybridomas were cloned by limiting dilution and by the soft agar technique according to a modification of the procedure of Coffino et al (19); mouse peritoneal macrophages containing 2.5 x 106 cells were prepared 48 h in advance of cloning and used as feeder layers for the cloned hybridomas.…”

Clonal evolution of myeloma cells leads to quantitative changes in immunoglobulin secretion and surface antigen expression.

“…Myeloma cells were cloned in soft agar by using a modification (15) of a method described by Coffino et al (24). Individual colonies were picked with a micropipette and expanded in liquid suspension cultures.…”

Antigen-binding mutants of mouse myeloma cells.

“…Although this enormous genetic instability in cultured myeloma cells might have suggested a somatic generation of antibody diversity, all of the mutations reported so far (8,12,13) Between 250 and 1000 clones were scored per plate. Subclones and presumptive variants were removed from the agarose and grown up to mass culture as described previously (17).…”