ROS production by PBL from SSc patients and healthy controls. (A) PBL from 17 healthy controls (white bar) and 34 SSc patients (black bar), isolated from peripheral blood were stained with 20 μM DCFH-DA for 20 minutes, and fluorescence was measured in a plate reader fluorimeter. 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 PBL. (B) ROS production by PBL from one healthy control (black line) and one SSc patient (grey line) was analyzed by FACS analysis. A representative histogram of three independent experiments is shown. (C and D) SSc CD14- (C) and SSc CD19- cells (D) were purified from PBL using CD14 and CD19 microbeads. The total fractions of cells (PBL, white bars) and the collected depleted fractions (black bars) were stained with 20 μM DCFH-DA for 20 minutes, and fluorescence was measured on a plate reader fluorimeter. Data are means ± SD of three independent experiments with cells from three distinct subjects. DCFH-DA, 2′, 7′-dichlorodihydrofluorescin diacetate; PBL, peripheral blood lymphocytes; ROS, reactive oxygen species; SSc, systemic sclerosis.