Cloning of mouse myeloma cells and detection of rare variants

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 and had an enhancing effect on colony formation. Immunoglobulin production by cloned 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 in these cultures was assessed by a Ouchterlony immunodiffusion of the supernatant medium, and by incubating the cells with radioactive amino acids and analyzing the intracellular and secreted immunoglobulin on polyacrylamide gels.