Fig. 2. Increase of the intracellular oxidant status by low concentrations of t-BHP or BSO without impairment of cell-cycle progression. (A) Intracellular pro-oxidant status was determined by fluorimetric detection of DCF. Values represent the increase in fluorescence recorded 30 minutes after addition of 0.1 µM t-BHP or 10 µM BSO, relative to the values recorded in control cells. Data are the mean (± s.d.) of triplicate wells from two experiments. (B) Cells were treated with 0.1 µM t-BHP (empty circles), 10 µM BSO (filled squares) or vehicle (filled circles) 48 hours after passage and simultaneously incubated with 10 µg/ml BrdU. Labelled cells were identified by immunocytochemistry at the indicated time points after treatment. Results represent the mean (± s.d.) of three replicate dishes from the third passage. (C) Whole cell lysates were prepared from cultures grown under control or pro-oxidant conditions during the fourth and fifth passage. Oxidised proteins were quantified as described in Materials and Methods. Results are the mean (± s.d.) of three replicate cultures. *P<0.05. (D) Intracellular pro-oxidant status was determined by flow cytometry in late passage cultures (>25 CPD) before (baseline) and 30 minutes after addition of 200 µM H₂O₂. Values are the mean of three replicate dishes.