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In all previous studies, bloodstream forms of Trypanosoma brucei could be grown in vitro only when supported by a feeder layer of mammalian fibroblasts. We have axenically cultivated bloodstream T. brucei by adding L-cysteine at regular intervals and appropriate concentrations. The optimum cysteine concentration depends on cell density and is close to physiological serum levels. At concentrations greater than 24 mg/liter (2 X 10(-4) M), cysteine was acutely toxic to trypanosome concentrations of 3 X 10(7)/ml. Toxicity was prevented by addition of pyruvate or catalase, which neutralize H2O2 produced by cysteine autoxidation. In uptake studies using [35S]cysteine and [35S]cystine, T. brucei efficiently incorporated only cysteine. The Km for cysteine uptake was 4 X 10(-4) M. Cystine supported axenic growth if low concentrations of 2-mercaptoethanol were added at regular intervals.
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