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A method is described for obtaining antibody-producing hybridomas that are preferentially retained in cultures of fused mouse spleen and myeloma cells. Hybridomas are produced by fusing mouse myeloma cells that are deficient in adenosine phosphoribosyltransferase (APRT) with mouse spleen cells containing Robertsonian 8.12 translocation chromosomes. The cell fusion mixtures are exposed to a culture medium that can be utilized only by APRT-positive cells, which results in the elimination of both unfused APRT-deficient myeloma cells and non-antibody-producing APRT-deficient hybridomas that arise by segregation of the 8.12 translocation chromosomes containing the APRT genes and the active heavy chain immunoglobulin gene.

Moloney lymphoma cells are larger than normal cells of mouse spleen, lymph-node, and thymus. Use of electronic cell-size anaylsis in conjunction with specific immunocytolysis permits difjerential counting of host and tumor cells. Increase of lymphoma cells occurs first in spleen and then in lymph nodes; in thymus it occurs only during the terminal stages of tumor growth.

A system for high-speed sorting of fluorescent cells was able to sort mouse spleen cells from Chinese hamster ovarian cells after development of fluorochromasia. Highly fluorescent fractions separated after similar treatment from mouse spleen cells immunized to sheep erythrocytes were enriched in antibody-producing cells by factors of 4 to 10.

A suspension of mouse spleen cells can be separated into two populations on the basis of their ability or inability to adhere to plastic dishes. It was found that both adherent and nonadherent cells were necessary for the induction of antibody formation to sheep red blood cells in vitro. Exposure of adherent cells to antigen for brief periods of time was sufficient to initiate a maximal in vitro response.

The activity of uroporphyrinogen III cosynthetase is much lower in hemolyzates from mature cattle with congenital erythropoietic porphyria than in hemolyzates from mature normal cattle. The porphyric hemolyzates do not inhibit the cosynthetase activity present in normal hemolyzates or in extracts of mouse spleen.

Populations of mouse spleen cells, without those cells that adhere to glass or plastic, exhibit little or no capacity for formation of antibody to sheep erythrocytes in the Mishell-Dutton culture system. When 5 x 10 -5 M mercaptoethanol is added to the culture medium, the antibody-forming capacity of these nonadherent spleen cells is restored to that of unfractionated spleen cells.

Depression of the in vitro immune response of mouse spleen cell suspensions to sheep erythrocytes by removal of macrophages can be reversed by the addition of supernatants from peritoneal macrophage cultures. Supernatant activity can be absorbed by the red cell antigen, and supernatant-treated red cells are stimulatory in the absence of macrophages or supernatant.