T lymphocyte colonies stimulated by different mitogens require diverse culture conditions.
Normal human peripheral blood mononuclear cells form colonies of T lymphocytes in a semi-solid agar matrix when stimulated by a variety of mitogens. In this report, we attempt to determine the optimal conditions for the formation of T lymphocyte colonies by cells stimulated with phytohemagglutinin (PHA), pokeweed mitogen (PWM), Concanavalin A (Con A), or Staphylococcal protein A (SPA). We conclude that optimal conditions differ for each mitogen used. Cultures stimulated by PWM or Con A showed a significant requirement for feeder layers composed of human peripheral blood mononuclear cells. Two-mercaptoethanol significantly enhanced the number of T-cell colonies when PWM, Con A, or SPA, but not PHA, were added as mitogens. Fetal calf serum (FCS) was required for optimal conditions when Con A or SPA but not PWM or PHA were used to stimulate mononuclear cells. Cells stimulated by PHA or PWM produced more T-cell colonies in a 2-step assay than a 1-step assay, whereas the reverse was true with Con A or SPA. Optimal cell concentrations, mitogen doses, and culture kinetics also differed for each mitogen used in the T-cell colony assay.
D002455:Cell Division / Q000187:drug effects*; D002478:Cells, Cultured; D003208:Concanavalin A / Q000276:immunology; D003470:Culture Media; D006801:Humans; D008213:Lymphocyte Activation; D008623:Mercaptoethanol / Q000494:pharmacology; D008934:Mitogens / Q000276:immunology*; D010835:Phytohemagglutinins / Q000276:immunology; D011043:Pokeweed Mitogens / Q000276:immunology; D013205:Staphylococcal Protein A / Q000276:immunology; D013601:T-Lymphocytes / Q000276:immunology*
D003470:Culture Media; D008934:Mitogens; D010835:Phytohemagglutinins; D011043:Pokeweed Mitogens; D013205:Staphylococcal Protein A; D003208:Concanavalin A; D008623:Mercaptoethanol