In-vitro NK-DC co-culture
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Whole blood samples were obtained from healthy laboratory donors for the isolation of purified and enriched NK and DC cell populations from PBMC using magnetic cell separation.
- The development of an NK-DC co-culture system required two independent steps.
- Firstly, DC were prepared using a pre-optimised magnetic cell sorting kit for the extraction of CD14+ monocytes from PBMC.
- Following isolation, CD14+ monocytes were treated with 100 ng/ml of GM-CSF and 1000 U/ml of IL-4 in RPMI containing 5% autologous serum and maintained in a 5% CO2 cell culture incubator at 37°C for 5 days.
- This altered their phenotype to that of immature dendritic cells (iDC) with a population of >90% CD14−CD1a+ iDC cells, comparable with the published literature +CD16+ NK cell.
- Purities of >98% were attained consistent with published literature 2 cell culture incubator at 37°C.
- Co-culture cell ratios…
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Whole blood samples were obtained from healthy laboratory donors for the isolation of purified and enriched NK and DC cell populations from PBMC using magnetic cell separation.
- The development of an NK-DC co-culture system required two independent steps.
- Firstly, DC were prepared using a pre-optimised magnetic cell sorting kit for the extraction of CD14+ monocytes from PBMC.
- Following isolation, CD14+ monocytes were treated with 100 ng/ml of GM-CSF and 1000 U/ml of IL-4 in RPMI containing 5% autologous serum and maintained in a 5% CO2 cell culture incubator at 37°C for 5 days.
- This altered their phenotype to that of immature dendritic cells (iDC) with a population of >90% CD14−CD1a+ iDC cells, comparable with the published literature +CD16+ NK cell.
- Purities of >98% were attained consistent with published literature 2 cell culture incubator at 37°C.
- Co-culture cell ratios of 1∶1 and 1∶5 (NK∶DC) were then studied as these ratios are most favourable for DC maturation 6 of each cell type in a final media volume of 500 µl.
- The control wells had either DC or NK cells in isolation.
- Trans-well experiments were performed using the same conditions, but in the presence or absence of a 0.4 µm insert to separate NK cells and DC.
- At the end of co-culture C were tested for the expression of the maturation markers C86 (, 2331 ) and HLA- and the chemokine receptor CC7 .
- The DC gate on flow cytometry was defined by a combination of scatter plot and CD56 staining to identify NK cells.
- The Δ Mean Fluorescence Intensity (Δ MFI) per experiment for CD86, HLA-DR and CCR7 was calculated as the difference in MFI for DC in co-culture with NK cells versus DC in isolation (background maturation).
- Corresponding isotype controls were used.
- Cells were analysed using a FACSCalibur flow cytometer with Winmdi 2.9 software , acquiring information from a total of 5,000 gated cells.
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Primary cell isolation and viral infection
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Bone marrow macrophages (BMM) and bone marrow dendritic cells (BMDC) were generated as described previously
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Immunoprotection analysis of Com1- and HspB-pulsed BMDCs
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Mouse bone marrow dendritic cells (BMDCs, CD11c+) were isolated from the bone marrow of BALB/c mice, according to the protocol described previously [2.
- Approximately 1 × 106 cells was added to each well of a six-well plate, and mouse GM-CSF (20 ng/ml) and IL-4 (10 ng/ml) was added to the culture medium every other day.
- After 6 days of culture, BMDCs were stimulated with C.
- burnetii I Ag (10 μg/ml), Com1 (10 μg/ml), HspB (10 μg/ml), E.
- coli LPS (2 μg/ml), or 25 μl elution buffer (mock pulse) for 24 h at 37°C and 5% CO2.
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mDCs inhibited Treg differentiation.
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D–F, Bone marrow precursor cells freshly isolated from naïve mice were cultured in vitro in the presence of GM-CSF and IL-4.
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T. crassiceps-inoculation induces elevation of IL-4 and hepatic expression of Ym-1.
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Six weeks post-inoculation, IL-4 and IFN-γ serum levels were determined from Tc-inoculated and non-inoculated mice by ELISA.
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Antibodies and proteins
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Antibodies directed against IκBα (#4814), phospho p38 (#9211), p38 (#9212), phospho ERK1/2 (#9106), ERK1/2 (#9102), phospho JNK1/2 (#9251), JNK1/2 (#9252) and phospho c-Jun (#3270) were all from Cell Signaling Technology.
- Wnt5a antibody was from Biotechnology (Sc-30224), NFATC2 for Western blotting was from and for immunofluorescence from (Sc-13034), and the mouse monoclonal β-actin from .
- LPS (from E.
- Recombinant IL-4 and IL-13 proteins were bought from .
- HlyA was obtained from Prof. S. Bhakdi (Mainz University, Germany).
- HlyA was preincubated with polymyxin B (50 µg/ml) at 4°C for 30 min to remove any possible LPS contamination.
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Functional assays
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For cytokine signalling, LN T cells (5 × 106 cells/ml) were cultured 16 h with recombinant mouse IL-7 (10 ng/ml), IL-4 (40 ng/ml), IL-6 (45 ng/ml& ) or IL-15 (100 ng/ml& ) as previously described (
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Immune modulation by IL-12 shRNA silenced DC.
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Co-cultured T cells were harvested and gene expression of IFNγ, IL-2, IL-4 and IL-10 were detected by RT-PCR.
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Spleen DC isolation and peripheral blood derived cultures
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After experimentation the following protocol, adapted from mammalian methods 2 flasks with phenolic caps at a density of approximately 5×106 cells/ml of supplemented medium'>L-15 medium (20% FBS, 50 µg/mL gentamicin, and 0.25 µg/mL fungizone).
- Cells were allowed to adhere for two hours, at which point non-adherent cells were washed off and new media was added containing mammalian IL-4 (250 U/ml, human:, ; :, , ) and GM-CSF (1000 U/ml, human:,; :).
- Cells were “fed” additional IL-4 and GM-CSF (same concentration as above) on days two, four, and six.
- Non-adherent cells removed at the 2 hr time point were transferred to a new flask for culture without addition of exogenous IL-4 or GM-CSF (or removal of non-adherent cells).
- These mixed cultures (sans cytokines) yielded much the same result as the adherent culture with cytokines.
- A more direct comparison of adherent cultures with or without exogenous cytokines was made.
- No discernable…
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After experimentation the following protocol, adapted from mammalian methods 2 flasks with phenolic caps at a density of approximately 5×106 cells/ml of supplemented medium'>L-15 medium (20% FBS, 50 µg/mL gentamicin, and 0.25 µg/mL fungizone).
- Cells were allowed to adhere for two hours, at which point non-adherent cells were washed off and new media was added containing mammalian IL-4 (250 U/ml, human:, ; :, , ) and GM-CSF (1000 U/ml, human:,; :).
- Cells were “fed” additional IL-4 and GM-CSF (same concentration as above) on days two, four, and six.
- Non-adherent cells removed at the 2 hr time point were transferred to a new flask for culture without addition of exogenous IL-4 or GM-CSF (or removal of non-adherent cells).
- These mixed cultures (sans cytokines) yielded much the same result as the adherent culture with cytokines.
- A more direct comparison of adherent cultures with or without exogenous cytokines was made.
- No discernable differences were seen (except perhaps small differences in the kinetics of the cultures), and addition of cytokines was subsequently abandoned.
- Cells were cultured at RT in the atmosphere.
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Progressive acquisition of CD200R correlates with effector cytokine secretion.
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Intracellular cytokine staining with CD200R expression is shown for TNFα, IL-2, IL-4 and IFNγ.
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