normal human dendritic cell  (Lonza)

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Serum miR-29a is upregulated in acute Graft versus Host Disease and activates dendritic cells through TLR binding

doi: 10.4049/jimmunol.1601778

Figure Lengend Snippet: (A) Human PBMCs (n=9) from healthy donors were treated with DOTAP-miR29a, DOTAP-miR-16, DOTAP alone for 48 hrs. T cell activation was measured by CD69 upregulation on CD3+ T cells by flow cytometry. (B) CD11c+ CD14- human dendritic cells (n=5) were treated with DOTAP-miR29a, DOTAP-miR-16, DOTAP alone for 48 hrs. DC maturation was assessed by mean fluorescence intensity (MFI) of CD40 and CD83 by FACS and (C) Detection of TNF-a and IL-6 in supernatants by ELISA. (D) Human dendritic cells were transfected with Flag TLR8 or Flag EV plasmids with or without Scr or miR-29a or miR-155. Immunoprecipitation was carried out using IgG or Flag Antibody and RNA-IP showing enrichment of only miR-29a in TLR8 transfected cells. Paired t-test was used to compare differences, p<0.05 was considered significant.

Article Snippet: PBMCs and normal human dendritic cells were purchased from ALLCELLS (Alameda, CA).

Techniques: Activation Assay, Flow Cytometry, Fluorescence, Enzyme-linked Immunosorbent Assay, Transfection, Immunoprecipitation

Journal: BMC Microbiology

Article Title: c-KIT signaling is targeted by pathogenic Yersinia to suppress the host immune response

doi: 10.1186/1471-2180-13-249

Figure Lengend Snippet: Differential response to Y. pestis infection in human dendritic cells correlates with naturally-expressed c-KIT levels. (A) Differential expression of c-KIT in human dendritic cells. NHDCs (20,000) from seven different donors (D1-7) were cultured in LGM-3 for 4 days. Both adherent and suspension cells were collected, fixed, labeled with (PE)-conjugated c-KIT (Ab81) antibody, and subjected to flow cytometry analysis. 10,000 cells were acquired to generate histograms and a bar graph (B) that depict fluorescence intensity distribution and mean channel fluorescence intensity. The control sample (C) was generated from a pool of unlabeled NHDC from the seven donors. (C) NHDCs that express high levels of c-KIT exhibit increased inhibition of TNF-α release upon Y. pestis infection. NHDCs from seven donors were cultured in LGM-3 for 4 days prior to treatment. Cells from a single donor were plated in 6 replicates (in a 24-well cluster dish): 2 wells were treated with LPS ( E. coli 055:B5, 5 μg/ml) and 4 wells received Y. pestis Ind195 at MOI 20. The inhibition of TNF-α production by Y. pestis -infected cells was determined relative to LPS-treated cells for each donor. The data presented was generated from an average of four replicates of Y. pestis -infected cells versus the average of two replicates treated with LPS. The ELISA for each experimental sample was performed in triplicate.

Article Snippet: Normal human dendritic cells (NHDC) (LONZA, Allendale, NJ) were cultured in LGM-3 Growth Medium (LONZA).

Techniques: Infection, Quantitative Proteomics, Cell Culture, Suspension, Labeling, Flow Cytometry, Fluorescence, Control, Generated, Inhibition, Enzyme-linked Immunosorbent Assay