title: Stimulant-free preculture in heterologous serum-supplemented medium induces unresponsiveness of T cells to subsequent mitogenic stimulation.
abstract: Quite often freshly isolated lymphocytes are kept in culture before experimentation for 1 or more days without any stimulus. Most of the time, culture is supplemented with fetal bovine serum (FBS) which is heterologous to all species except bovine. In the present study, we found that freshly isolated murine T cells show a good proliferative response to concanavalin A (Con A) and phorbol ester (PMA)/ionomycin in FBS medium, without any detectable background proliferation. However, the cells kept in the same culture without any stimulus for prolonged period of time (referred to as preculture in this report) showed reduced response to Con A and PMA/ionomycin in a time-dependent manner. Almost a complete loss of response to Con A was observed within 1 day of preculture. However, loss of response to PMA/ionomycin was observed only after 2 days of preculture. Interestingly, similar preculture in autologous mouse serum-supplemented media did not cause any loss of the response to these mitogens. The loss of responsiveness of T cells during preculture in heterologous serum was irreversible. The heterologous serum-induced unresponsiveness of T cells to these mitogens was also prevented by adding Calphostin C, a specific protein kinase C (PKC) inhibitor, during preculture in heterologous serum. These results showed that prolonged stimulant-free preculture in heterologous serum induces irreversible unresponsiveness of T cells to mitogens through the down regulation of T cell receptor signaling pathway, which can be prevented by autologous serum or a PKC inhibitor.
authors.affiliation: Molecular Biology Unit, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221005, India.
authors.forename: S
authors.initials: S
authors.lastname: Kumar
authors.forename: R
authors.initials: R
authors.lastname: Chakrabarti
mesh_terms: D000818:Animals; D015478:Antigens, Heterophile / Q000276:immunology*; D001798:Blood Proteins / Q000276:immunology; D002417:Cattle; D002478:Cells, Cultured; D003470:Culture Media; D008213:Lymphocyte Activation / Q000187:drug effects; D051379:Mice; D008807:Mice, Inbred BALB C; D008934:Mitogens / Q000494:pharmacology; D013601:T-Lymphocytes / Q000166:cytology* / Q000276:immunology*
chemical_list: D015478:Antigens, Heterophile; D001798:Blood Proteins; D003470:Culture Media; D008934:Mitogens