ABSTRACT
Since it is difficult to study human thymocyte maturation in vitro, we have developed an in vitro thymocyte culture system which has allowed us to select the optimal growth conditions for thymocyte subpopulations. Three thymocyte subpopulations (CD3-CD1-, CD1+CD3-, and CD3+CD1-) were isolated by a single step percoll density gradient centrifugation and indirect panning procedure using anti-CD1 and anti-CD3 monoclonal antibodies, and their purity was checked by flow cytometry. The combination of concanavalin A (Con A), tetradecanoylphorbol acetate (TPA), and IL-2 was shown to be the most reliable stimulus for the proliferation of CD3-CD1- thymocytes for up to 15 days in a culture system in vitro. Flow cytometric analysis for the phenotypic change of CD3-CD1- thymocytes revealed a steady increase of CD3 antigen after a 3-day cultivation, whereas there was no change in CD1 antigen intensity. A combination of Con A and IL-2 was both sufficient and necessary to induce growth of CD3+CD1- thymocytes. The major population of immature cortical thymocytes (CD3-CD1+ or CD3+CD1+), which are considered to be the most unresponsive dead-end cells, could not be maintained or stimulated with any combination used in this experiment, even in the presence of thymic accessory cells.
Subject(s)
Humans , Infant , Infant, Newborn , Antigens, CD , Antigens, CD1 , CD3 Complex , Antigens, Differentiation, T-Lymphocyte , Cell Cycle , Cell Division/drug effects , Cells, Cultured , Ionophores/pharmacology , Lymphocyte Activation/drug effects , Receptors, Antigen, T-Cell , T-Lymphocytes/cytology , Tetradecanoylphorbol Acetate/pharmacologyABSTRACT
Intact and gossypol-treated spermatozoa from healthy man were labeled withCon A-HRP-G two-step method.Comparative observations of these sperms were doneunder LM,TEM and SEM.Results show that there are gold particles on spermsurface membrane.Their distribution is more dens on plasma membrane of spermhead and middle piece.On the intracellular membrane structures of sperm which plasmamembrane was damaged by gossypol the gold particles also can be seen,especially onthe membranes of acrosome and mitochondria.It was proved that Con A receptorsare present not only on plasma membrane of sperm,but also on the membrane ofacrosome and mitochondrion.The significance of Con A receptors on sperm struc-tures,advantages of Con A-HRP-G two-step labeled method and comparativeobservations under LM,TEM and SEM were discussed.