ROS production by T cells from SSc patients and healthy controls. (A) T lymphocytes from 12 healthy controls (white bar) and 23 SSc patients (black bar) purified using a negative selection procedure were stained with 20 μM DCFH-DA, and the fluorescence was quantified in a plate reader fluorimeter (upper panel). Each patient and control was tested three times and the mean value used to calculate the mean of each group. Data are means ± standard deviation (SD). *
P <0.05 compared to normal T cells. Representative histogram of ROS production in T cells from one healthy control (black line) and one SSc patient (grey line) analyzed by FACS analysis is shown in the lower panel. (B) ROS production in total (grey line) and in CD3+ PBL (black line) from one SSc patient simultaneously stained with 2 μM DCFH-DA and monoclonal PerCP-conjugated anti-CD3 antibody for 20 minutes was analyzed by FACS analysis. Histogram is representative of three independent experiments. (C) CD3+ T lymphocytes isolated from 10 SSc patients and treated with NAC (10 mM, 1 hour) were stained with 20 μM DCFH-DA for 20 minutes, and the fluorescence was measured in a plate reader fluorimeter. Each treatment was tested three times and the mean value used to calculate the mean of each group. Data are means ± SD. *
P <0.05 compared to untreated T cells (control). (D) Untreated (control, grey line) or treated with NAC CD3+ T cells (black line) were simultaneously stained with 2 μM DCFH-DA and monoclonal PerCP-conjugated anti-CD3 antibody for 20 minutes, and ROS production was analyzed by FACS analysis. Representative histogram of three independent experiments is shown. DCFH-DA, 2′, 7′-dichlorodihydrofluorescin diacetate; NAC, N-acetylcysteine; PBL, peripheral blood lymphocytes; PerCP, peridinin chlorophyl protein; ROS, reactive oxygen species; SSc, systemic sclerosis.