Isolation of hybridoma forming cells from immune spleens by unit gravity sedimentation.
In 1975 a method for the production of hybridomas that secrete monoclonal antibodies was first described (1). Since that time, surprisingly little information has emerged on the identity of the fusible spleen cell. In this report we describe the isolation of hybridoma forming cells from immune spleens and the enrichment of B cell populations in different states of activation. The B cells that reside in immune spleens are comprised of heterogeneous populations that differ in their states of activation. These populations have been difficult to separate and, therefore, difficult to characterize. Two factors that discriminate B cells in different states of activation are cell size and density. Unit gravity sedimentation is a simple, reliable and reproducible method for separating cells based on their size and density. Immune B cells were fractionated on a 10 to 25% serum gradient at unit gravity. Cells were collected and pooled, when necessary, to perform functional assays. Fractionated B cells were assayed for hybridoma formation, PFC response, Ig secretion and proliferation. The cells that exhibited hybridoma formation, PFC activity and Ig secretion were all found in the large cell fractions that comprised less than 10% of the cells recovered from the gradient. Proliferating B cells were found primarily in smaller cell fractions compared to antibody forming cells. Resting B cells were located in the smallest cell fractions recovered from the gradient. Data show that separation of immune B cells by unit gravity sedimentation is an effective means to isolate hybridoma forming cells and enrich for B cell subpopulations in different states of activation.
Hybritech Incorporated, San Diego, California 92196-9006.
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Kuus-Reichel
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Beebe
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Knott
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis; D001402:B-Lymphocytes / Q000378:metabolism; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D006825:Hybridomas; D007136:Immunoglobulins / Q000032:analysis / Q000096:biosynthesis; D051379:Mice; D008807:Mice, Inbred BALB C; D013154:Spleen / Q000166:cytology / Q000276:immunology*
D000911:Antibodies, Monoclonal; D007136:Immunoglobulins