Journal: Arthritis Research & Therapy

Article Title: Intracellular free radical production by peripheral blood T lymphocytes from patients with systemic sclerosis: role of NADPH oxidase and ERK1/2

doi: 10.1186/s13075-015-0591-8

Figure Lengend Snippet: 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.

Article Snippet: Cells were then stained with 20 μM DCFH-DA for 20 minutes and analyzed in a plate reader fluorimeter (Wallac, Becton Dickinson).

Techniques: Isolation, Staining, Fluorescence, Control, Standard Deviation, Purification

Journal: Arthritis Research & Therapy

Article Title: Intracellular free radical production by peripheral blood T lymphocytes from patients with systemic sclerosis: role of NADPH oxidase and ERK1/2

doi: 10.1186/s13075-015-0591-8

Figure Lengend Snippet: 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.

Article Snippet: Cells were then stained with 20 μM DCFH-DA for 20 minutes and analyzed in a plate reader fluorimeter (Wallac, Becton Dickinson).

Techniques: Purification, Selection, Staining, Fluorescence, Control, Standard Deviation, Isolation

Journal: Arthritis Research & Therapy

Article Title: Intracellular free radical production by peripheral blood T lymphocytes from patients with systemic sclerosis: role of NADPH oxidase and ERK1/2

doi: 10.1186/s13075-015-0591-8

Figure Lengend Snippet: Role of NOX2 in ROS production by SSc T cells. (A) A representative gp91phox immunoblot of three independent experiments is shown. T cells were isolated from healthy controls (N) and SSc patients (SSc) and then analyzed by immunoblotting with specific antibodies. The lower panel shows the densitometric analysis from distinct experiments with five healthy controls and five SSc patients. Data are means ± standard deviation (SD). * P <0.05 compared to normal T cells. (B) Real time-PCR for gp91phox expression in T cells was isolated from five healthy controls and five SSc patients. Each patient and control 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 normal T cells. (C) T cells isolated from five SSc patients were treated with DPI (20 μM, 1 hour), stained with 20 μM DCFH-DA and subsequently analyzed 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) ROS production in SSc T lymphocytes transiently transfected with control siRNA (CTRL siRNA) or gp91phox targeted siRNA (gp91phox siRNA) for 72 hours (upper panel). Data are means ± SD of three independent experiments with cells from three distinct patients. * P <0.05 compared to control siRNA. Transfection efficiency was monitored by western blot (lower panel). DCFH-DA, 2′, 7′-dichlorodihydrofluorescin diacetate; NOX2, NADPH oxidase 2; ROS, reactive oxygen species; siRNA, small interfering RNA; SSc, systemic sclerosis.

Article Snippet: Cells were then stained with 20 μM DCFH-DA for 20 minutes and analyzed in a plate reader fluorimeter (Wallac, Becton Dickinson).

Techniques: Western Blot, Isolation, Standard Deviation, Real-time Polymerase Chain Reaction, Expressing, Control, Staining, Transfection, Small Interfering RNA

Journal: Arthritis Research & Therapy

Article Title: Intracellular free radical production by peripheral blood T lymphocytes from patients with systemic sclerosis: role of NADPH oxidase and ERK1/2

doi: 10.1186/s13075-015-0591-8

Figure Lengend Snippet: pERK1/2 implication in ROS production. (A) T lymphocytes from five SSc patients were incubated with PD98059 (50 μM, 2 hours) or left untreated. After staining with 20 μM DCFH-DA, fluorescence was analyzed in a plate reader fluorimeter (left panel). Each treatment 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 untreated T cells (control). Representative histogram of ROS production by SSc T cells is shown in the right panel. Untreated (control, grey line) and PD98059 treated cells (black line) were stained with 2 μM DCFH-DA and analyzed by FACS analysis. (B) Representative pERK1/2 immunoblot of three independent experiments is shown (upper panel). T lymphocytes were isolated from five healthy controls and five SSc patients, treated with NAC (10 mM, 1 hour) or DPI (20 μM, 1 hour), and then analyzed by immunoblotting with specific antibodies. The lower panel shows the densitometric analysis from distinct experiments with five healthy controls and five SSc patients. Data are means ± SD. * P <0.05 compared to untreated normal T cells. ** P < 0.05 compared to untreated SSc T cells. (C) Representative Ha-Ras immunoblot of three independent experiments is shown (upper panel). T lymphocytes were isolated from five healthy controls and five SSc patients, treated with NAC (10 mM, 1 hour) or DPI (20 μM, 1 hour), and then analyzed by immunoblotting with specific antibodies. The lower panel shows the densitometric analysis from distinct experiments with five healthy controls and five SSc patients. Data are means ± SD. * P <0.05 compared to untreated normal T cells. ** P <0.05 compared to untreated SSc T cells. DCFH-DA, 2′, 7′-dichlorodihydrofluorescin diacetate; DPI, diphenylene iodonium; NAC, N-acetylcysteine; ROS, reactive oxygen species; SSc, systemic sclerosis.

Article Snippet: Cells were then stained with 20 μM DCFH-DA for 20 minutes and analyzed in a plate reader fluorimeter (Wallac, Becton Dickinson).

Techniques: Incubation, Staining, Fluorescence, Standard Deviation, Control, Western Blot, Isolation

Journal: Arthritis Research & Therapy

Article Title: Intracellular free radical production by peripheral blood T lymphocytes from patients with systemic sclerosis: role of NADPH oxidase and ERK1/2

doi: 10.1186/s13075-015-0591-8

Figure Lengend Snippet: Modulation of ROS production by cytokines. ROS production by T cells isolated from three healthy controls (A) and three SSc patients (B) and treated with IL-1 (10 ng/ml), IL-4 (15 ng/ml), IL-6 (5 ng/ml), IL-13 (50 ng/ml), IFNα (0.5 ng/ml), TNFα (1 ng/ml), TGFβ (1 ng/ml), and PDGF-BB (15 ng/ml) for 15 minutes. Cells were then stained with 20 μM DCFH-DA and analyzed in a plate reader fluorimeter. Data are means ± standard deviation (SD) of three independent experiments with cells from three distinct subjects. * P <0.05 compared to untreated T cells. DCFH-DA, 2′, 7′-dichlorodihydrofluorescin diacetate; IFNα, interferon alpha; IL, interleukin; PDGF-BB, platelet-derived growth factor beta; ROS, reactive oxygen species; SSc, systemic sclerosis; Th, T helper; TGFβ, transforming growth factor beta; TNFα, tumor necrosis factor alpha.

Article Snippet: Cells were then stained with 20 μM DCFH-DA for 20 minutes and analyzed in a plate reader fluorimeter (Wallac, Becton Dickinson).

Techniques: Isolation, Staining, Standard Deviation, Derivative Assay

Journal: Arthritis Research & Therapy

Article Title: Intracellular free radical production by peripheral blood T lymphocytes from patients with systemic sclerosis: role of NADPH oxidase and ERK1/2

doi: 10.1186/s13075-015-0591-8

Figure Lengend Snippet: Modulation of proliferation and cytokine production by ROS in activated T cells. (A) T cells from three healthy controls and three SSc patients were activated with CD3/CD28 magnetic beads, treated with DPI (20 μM, 1 hour) and pulsed with BrdU for 6 hours. Proliferation was measured with a colorimetric immunoassay in a plate reader fluorimeter. Data are means ± standard deviation (SD) of fold increase of SSc/N proliferation ratio. * P <0.05. (B) One representative flow cytometry analysis of IFNγ-producing T cells is shown. Normal T cells (upper panels) or SSc T cells (lower panels) were activated with CD3/CD28 magnetic beads and treated with DPI (20 μM, 1 hour). Plots represent live cells gated for CD4+ cells. Percentage of IFNγ-producing T cells in three healthy controls and three SSc patients are presented (right panel). Data are means ± SD. * P <0.05 compared to normal untreated T cells. ** P <0.05 compared to SSc untreated T cells. (C) One representative flow cytometry analysis of IL-4-producing T cells is shown. Normal T cells (upper panel) or SSc T cells (lower panel) were activated with CD3/CD28 magnetic beads and treated with DPI (20 μM, 1 hour). Plots represent live cells gated for CD4+ cells. Percentage of IL-4-producing T cells in three healthy controls and three SSc patients are presented (right panel). Data are means ± SD. * P <0.05 compared to normal untreated T cells. ** P <0.05 compared to SSc untreated T cells. BrdU, bromodeoxyuridine; DPI, diphenylene iodonium; IFN, interferon; PMA, phorbol myristate acetate; ROS, reactive oxygen species; SSc, systemic sclerosis.

Article Snippet: Cells were then stained with 20 μM DCFH-DA for 20 minutes and analyzed in a plate reader fluorimeter (Wallac, Becton Dickinson).

Techniques: Magnetic Beads, Standard Deviation, Flow Cytometry