Journal:

Article Title: Calprotectin Expression by Gingival Epithelial Cells

doi: 10.1128/IAI.69.5.3248-3254.2001

Figure Lengend Snippet: Treatment of immortalized keratinocytes with IL-1β (0.1, 1, and 10 ng/ml) for 2 or 4 h. Immortalized keratinocytes were incubated with various concentrations of IL-1β (0.1, 1, and 10 ng/ml) for 2 h (black bars) or 4 h (crosshatched bars). Expression of mRNA for IL-8-, MRP8-, and MRP14-specific mRNA was determined by RNase protection assay. The signal intensity for the protected fragments was normalized to the abundance of the internal control GAPDH. The data shown are the mean ± the standard deviation (n = 3).

Article Snippet: Immortalized keratinocytes were stimulated by incubation with lipopolysaccharide from E. coli O55:B5 (Sigma, St. Louis, Mo.), phorbol 12-myristate 13-acetate (PMA; Sigma) or recombinant human IL-1β (R&D Systems, Inc., St. Paul, Minn.).

Techniques: Incubation, Expressing, Rnase Protection Assay, Standard Deviation

Journal:

Article Title: Adeno-Associated Virus Type 2-Mediated Transduction of Human Monocyte-Derived Dendritic Cells: Implications for Ex Vivo Immunotherapy

doi: 10.1128/JVI.75.19.9493-9501.2001

Figure Lengend Snippet: Luciferase activity in control and rAAV-transduced MO cultures. Freshly isolated MO were either mock transduced or transduced with rAAV (100 MOI). Following transduction, monocytes were either cultured without cytokines (Mono) or with GM-CSF alone (GM) or with IL-4 plus GM-CSF (IL-4+GM). Analysis of luciferase activity (RLU) was performed 2, 4, and 7 days postinfection.

Article Snippet: To generate DC, MO were cultured in RPMI 1640 medium with 2 mM l -glutamine (Mediatech), 10% fetal calf serum (HyClone), human recombinant interleukin 4 (IL-4) (17 ng/ml; R&D Systems), and 800 U of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) (Leukine, Immunex) per ml for up to 10 days, with partial cytokine medium replacement every 2 to 3 days.

Techniques: Luciferase, Activity Assay, Isolation, Transduction, Cell Culture

Journal:

Article Title: Adeno-Associated Virus Type 2-Mediated Transduction of Human Monocyte-Derived Dendritic Cells: Implications for Ex Vivo Immunotherapy

doi: 10.1128/JVI.75.19.9493-9501.2001

Figure Lengend Snippet: Luciferase activity in fluorescence-activated cell-sorted CD40- and CD1a-positive DCs generated from MO transduced with rAAV-luc. Freshly adherent MO from human peripheral blood were transduced with rAAV-luc (100 MOI) and subsequently cultured with IL-4 plus GM-CSF for 7 or 10 days. Cells were then sorted using fluorescent antibodies to CD40 and CD1a into populations that were either double-positive (CD40+ CD1a+) or single-positive (CD1a+ or CD40+). Less than 5% of harvested cells were negative for both CD1a and CD40. Luciferase activity (RLU) was expressed per microgram protein content in each lysate.

Article Snippet: To generate DC, MO were cultured in RPMI 1640 medium with 2 mM l -glutamine (Mediatech), 10% fetal calf serum (HyClone), human recombinant interleukin 4 (IL-4) (17 ng/ml; R&D Systems), and 800 U of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) (Leukine, Immunex) per ml for up to 10 days, with partial cytokine medium replacement every 2 to 3 days.

Techniques: Luciferase, Activity Assay, Fluorescence, Generated, Transduction, Cell Culture

Journal:

Article Title: Adeno-Associated Virus Type 2-Mediated Transduction of Human Monocyte-Derived Dendritic Cells: Implications for Ex Vivo Immunotherapy

doi: 10.1128/JVI.75.19.9493-9501.2001

Figure Lengend Snippet: Allogeneic MLR assays of AAV-luc transduced DCs. MO transduced with AAV on day 0 and cultured for 6 days in IL-4 and GM-CSF to generate DCs (A) or DCs transduced with AAV after 7 days cytokine culture and recultured with cytokines for 3 additional days (B) were used as stimulators in allogenic MLR assays. In each experiment, control stimulators included mock-transduced DCs as well as fresh autologous peripheral blood mononuclear cells (PBMC). Stimulators were irradiated and added at various numbers to 96-well culture plates. Fresh peripheral blood lymphocytes from donors unrelated to the DC donors were added to the irradiated stimulators at 100,000 cells/well, incubated for 5 days, and then pulsed with 3H-thymidine overnight prior to harvesting.

Article Snippet: To generate DC, MO were cultured in RPMI 1640 medium with 2 mM l -glutamine (Mediatech), 10% fetal calf serum (HyClone), human recombinant interleukin 4 (IL-4) (17 ng/ml; R&D Systems), and 800 U of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) (Leukine, Immunex) per ml for up to 10 days, with partial cytokine medium replacement every 2 to 3 days.

Techniques: Transduction, Cell Culture, Irradiation, Incubation

Journal:

Article Title: Adeno-Associated Virus Type 2-Mediated Transduction of Human Monocyte-Derived Dendritic Cells: Implications for Ex Vivo Immunotherapy

doi: 10.1128/JVI.75.19.9493-9501.2001

Figure Lengend Snippet: FISH analyses of AAV genome in DCs derived in culture from rAAV-luc-transduced MO. Peripheral blood adherent MO were either mock transduced or transduced with AAV-luc and subsequently cultured with IL-4 and GM-CSF for 10 days. DCs were fluorescence-activated cell sorted for CD40 and CD1a expression, and double-positive cells were used to prepare nuclei for FISH analysis by staining with a digoxigenin-labeled luciferase probe. (A) Representative field from mock-transduced cells. (B) Representative field from rAAV-luc-transduced cells, with arrows indicating signal from AAV proviral genome. Magnification, ×100.

Article Snippet: To generate DC, MO were cultured in RPMI 1640 medium with 2 mM l -glutamine (Mediatech), 10% fetal calf serum (HyClone), human recombinant interleukin 4 (IL-4) (17 ng/ml; R&D Systems), and 800 U of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) (Leukine, Immunex) per ml for up to 10 days, with partial cytokine medium replacement every 2 to 3 days.

Techniques: Derivative Assay, Transduction, Cell Culture, Fluorescence, Expressing, Staining, Labeling, Luciferase