Journal: The Journal of Biological Chemistry

Article Title: Human Memory, but Not Naive, CD4 + T Cells Expressing Transcription Factor T-bet Might Drive Rapid Cytokine Production *

doi: 10.1074/jbc.M114.608745

Figure Lengend Snippet: Kinetics of cytokine secretion by activated naive and memory CD4+ T cells. Naive and memory CD4+ T cells were isolated from CD4+ T cells by MACS microbeads, and the purity of naive or memory CD4+ T cells was more than 97% as determined by flow cytometry (a). Naive and memory CD4+ T cells were stimulated with (open histogram) or without (shaded histogram) PMA and ionomycin in the presence of BFA for 6 h. Cells were stained by intracellular staining and analyzed by FACS (b). Purified naive and memory CD4+ T cells were stimulated with immobilized anti-CD3 (1 μg/ml) and anti-CD28 (1 μg/ml) for 0 to 72 h, and the concentrations of cytokines IFN-γ, IL-2, and TNF-α were detected by ELISA (c). Data are representative of three separate experiments with similar results.

Article Snippet: Human ELISA kits for cytokines IFN-γ, IL-2, and TNF-α were purchased from BD Biosciences.

Techniques: Isolation, Flow Cytometry, Staining, Purification, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Altered effector CD4 + T cell function in IL-21R −/− CD4 + T cell-mediated graft-versus-host disease 1

doi: 10.4049/jimmunol.0902217

Figure Lengend Snippet: Cytokine production by splenic CD4+ T cells before and after transplantation. (A) Absolute number of donor H-2Kd-negative CD4+ T cells in the spleen. The number of donor CD4+ T cells was determined by multiplying the number of splenocytes by the percentage of H-2Kd-negative CD4+ T cells. Each dot depicts the number of donor CD4+ T cells in a mouse. Lines in the middle of dots indicate the average. Total mice analyzed were 15 recipients of wild type CD4+ T cells and 12 recipients of IL-21R−/− CD4+ T cells. (B) Intracellular staining of splenocytes after anti-CD3/CD28 stimulation. Splenocytes (1 × 106) were stimulated with anti-CD3/CD28 antibodies for 5–6 hours and stained with either anti-IFN-γ or anti-TNF-α antibody in combination with anti-CD4 antibody. A total of three recipients in each group were analyzed and a representative result is shown. (C) Cytokine production by CD4+ T cells in vitro. At day 14 or 21 after transplantation, splenic CD4+ T cells (5 × 105) were purified and stimulated with anti-CD3/CD28 antibodies for 18 hours. Concentrations of cytokines in the supernatants were determined by ELISA. Twelve mice were analyzed in each group after transplantation. Total mice analyzed before transplantation were 5–6 wild type and 8–9 IL-21R−/− mice. Asterisks denote statistical significance (p<0.05).

Article Snippet: ELISA kits for IL-2, IL-4, and IFN-γ were from BD Biosciences and ELISA kits for IL-21, IL-17, TNF-α, and TGF-β1 were from R&D Systems (Minneapolis, MN).

Techniques: Transplantation Assay, Staining, In Vitro, Purification, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Altered effector CD4 + T cell function in IL-21R −/− CD4 + T cell-mediated graft-versus-host disease 1

doi: 10.4049/jimmunol.0902217

Figure Lengend Snippet: CD4 allo-reaction in vitro was impaired after transplantation, and this impairment was not restored by CD25+ T cell depletion. (A) At day 14 after transplantation, 1 × 105 sorter-purified splenic CD4+ or CD25-negative CD4+ T cells (>98% purity) were cultured with 4 × 105 irradiated allogeneic C57BL/6-DBA2-F1 splenocytes for 4 days. The cells were pulsed with 1 μCi of [3H]-thymidine for the last 24 hours. Relative thymidine uptake to the value of wild type CD4+ T cells is depicted. (B) Culture was the same as in (A), but IFN-γ concentrations in the supernatants were determined by ELISA. (C) Sorter-purified splenic CD4+ or CD25-negative CD4+ cells from non-transplanted mice were cultured with irradiated allogeneic C57BL/6-DBA2-F1 splenocytes. Relative thymidine uptake to the number of wild type CD4+ T cells is depicted. Asterisks denote statistical significance (p<0.05).

Article Snippet: ELISA kits for IL-2, IL-4, and IFN-γ were from BD Biosciences and ELISA kits for IL-21, IL-17, TNF-α, and TGF-β1 were from R&D Systems (Minneapolis, MN).

Techniques: In Vitro, Transplantation Assay, Purification, Cell Culture, Irradiation, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Nanomedicine

Article Title: Toxicity evaluation of Gd 2 O 3 @SiO 2 nanoparticles prepared by laser ablation in liquid as MRI contrast agents in vivo

doi: 10.2147/IJN.S66164

Figure Lengend Snippet: ( A – B ) Cell viability and apoptosis in vitro on the Gd-NPs and ( D – E ) toxicity data of adaptive immunity were measured by flow cytometry 48 hours after injection in BALB/c mice (15 μmol/kg, mean ± SD, n=5). Notes: ( A ) Cell viability of L929 cells incubated with different concentrations (10 μM, 1 μM, and 100 nM) of the Gd-NPs for 24 and 48 hours. ( B ) Apoptosis rates of Raw264.7 cells were measured by flow cytometry at 48 hours after incubation of PBS, Gd-DTPA (500 nM), and the Gd-NPs (10 μM, 1 μM, 500 nM, and 100 nM). Cells were stained by Annexin V and PI. ( C ) Toxicity on innate immunity was measured by flow cytometry at 48 hours after injection in BALB/c mice (15 μmol/kg, mean ± SD, n=5). Here, 15 male BALB/c mice (6–8 weeks old) were divided randomly into three groups: 1) PBS (negative control, 100 μL), 2) Gd-DTPA (15 μmol/kg), and 3) Gd-NPs (15 μmol/kg). The expression levels of CD11b, CD206, CD40, and CD80/CD86 in monocytes/macrophages of peripheral blood, were detected by flow cytometry, respectively. ( D ) Concentrations of IL-1β, IL-2, and IL-4 in the serum. ( E ) Expression levels of CD69 in the lymphocyte cells of peripheral blood and lymph node, and ROS of neutrophils in peripheral blood, respectively. * P <0.05 compared with Gd-DTPA group. Abbreviations: BL, blood; DTPA, diethylenetriaminepentaacetic acid; IL, interleukin; LN, lymph node; MFI, mean fluorescence intensity; NP, nanoparticle; PBS, phosphate buffered saline; PI, propidium iodide; ROS, reactive oxygen species; SD, standard deviation.

Article Snippet: The serum was transferred into new Eppendorf tubes and assayed by IL-1β, IL-2, and IL-4 ELISA (enzyme-linked immunosorbent assay) kits (RayBiotech, Inc., Norcross, GA, USA) according to the provided instructions.

Techniques: In Vitro, Flow Cytometry, Injection, Incubation, Staining, Negative Control, Expressing, Fluorescence, Standard Deviation