Phenotype of clinical grade LAKs and DCs.
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DCs were generated by culture of CD14+ PBMCs with GMCSF and IL-4 for 5 days.
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ICI 182,780 revert E2-inhibitory effect in the release of a-defensins 1-3 by DCs.
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CD14+ cells isolated from fresh blood were cultured for 5 days in the presence of IL-4 and GM-CSF to differentiate into DCs.
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Isolation and culture of cells
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The adherence monocytes were cultured with RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS, , ), 50 ng/ml human IL-4 , and 50 ng/ml human granulocyte-macrophage colony-stimulating factor (GM-CSF) at 37°C and 5% CO2.
- IL-4 and GM-CSF were added to the cultured cells every other day.
- After 5 days of culture, more than 95% of the cells had converted to immature HMDCs (iHMDCs), with a phenotype of CD11c+, CD54 low, CD83 low, CD40 low, CD58 low, CD86low, and CD80low, as determined by flow cytometry.
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LMWHA inhibits IL-4 or IL-13-induced fibrocyte differentiation.
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Human PBMC were cultured in SFM, SFM with 1 ng/ml IL-4, SFM with 300 µg/ml LMWHA, or SFM with 1 ng/ml IL-4 and 300 µg/ml LMWHA, or SFM, SFM with 1 ng/ml IL-13, or SFM with 1 ng/ml IL-13 and 300 µg/ml LMWHA.
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Generation of expanded DCs
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Conventional DCs were obtained from peripheral blood CD14+ cells as described previously, with minor modification.
- Briefly, CD14+ cells were cultured under GM/IL-4 (100 ng/ml GM-CSF and 50 ng/ml IL-4) in medium'>IMDM medium containing 10% FBS and 1% penicillin and streptomycin.
- CD14+ monocytes were obtained by negative selection.
- Cells were washed in PBS, and lineage antigen-positive (CD2, CD3, CD19, CD20, CD56, CD66b, CD123, Glycophorin A) cells were removed by using the EasySep human monocyte enrichment kit without CD16 depletion .
- These lineage-negative CD14+ cells were cultured under 100 ng/ml human GM-CSF and 50 ng/ml human IL-4 in IMDM medium.
- Each cytokine was dissolved in PBS containing 0.1% BSA in 100 times the density used.
- For expansion, CD34+ cells were cultured under GM/SCF (100 ng/ml GM-CSF and 50 ng/ml SCF) or GM/IL-4 (100 ng/ml GM-CSF and 50 ng/ml IL-4) in IMDM medium.
- The medium was…
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Conventional DCs were obtained from peripheral blood CD14+ cells as described previously, with minor modification.
- Briefly, CD14+ cells were cultured under GM/IL-4 (100 ng/ml GM-CSF and 50 ng/ml IL-4) in medium'>IMDM medium containing 10% FBS and 1% penicillin and streptomycin.
- CD14+ monocytes were obtained by negative selection.
- Cells were washed in PBS, and lineage antigen-positive (CD2, CD3, CD19, CD20, CD56, CD66b, CD123, Glycophorin A) cells were removed by using the EasySep human monocyte enrichment kit without CD16 depletion .
- These lineage-negative CD14+ cells were cultured under 100 ng/ml human GM-CSF and 50 ng/ml human IL-4 in IMDM medium.
- Each cytokine was dissolved in PBS containing 0.1% BSA in 100 times the density used.
- For expansion, CD34+ cells were cultured under GM/SCF (100 ng/ml GM-CSF and 50 ng/ml SCF) or GM/IL-4 (100 ng/ml GM-CSF and 50 ng/ml IL-4) in IMDM medium.
- The medium was exchanged every 3 or 4 days.
- Cells were cultured in the MPC treatment plate (MD6 with Lid Low-Cell Binding; Nalge Nunc, ).
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Generation of expanded DCs
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Conventional DCs were obtained from peripheral blood CD14+ cells as described previously, with minor modification.
- Briefly, CD14+ cells were cultured under GM/IL-4 (100 ng/ml GM-CSF and 50 ng/ml IL-4) in medium'>IMDM medium containing 10% FBS and 1% penicillin and streptomycin.
- CD14+ monocytes were obtained by negative selection.
- Cells were washed in PBS, and lineage antigen-positive (CD2, CD3, CD19, CD20, CD56, CD66b, CD123, Glycophorin A) cells were removed by using the EasySep human monocyte enrichment kit without CD16 depletion .
- These lineage-negative CD14+ cells were cultured under 100 ng/ml human GM-CSF and 50 ng/ml human IL-4 in IMDM medium.
- Each cytokine was dissolved in PBS containing 0.1% BSA in 100 times the density used.
- For expansion, CD34+ cells were cultured under GM/SCF (100 ng/ml GM-CSF and 50 ng/ml SCF) or GM/IL-4 (100 ng/ml GM-CSF and 50 ng/ml IL-4) in IMDM medium.
- The medium was…
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Conventional DCs were obtained from peripheral blood CD14+ cells as described previously, with minor modification.
- Briefly, CD14+ cells were cultured under GM/IL-4 (100 ng/ml GM-CSF and 50 ng/ml IL-4) in medium'>IMDM medium containing 10% FBS and 1% penicillin and streptomycin.
- CD14+ monocytes were obtained by negative selection.
- Cells were washed in PBS, and lineage antigen-positive (CD2, CD3, CD19, CD20, CD56, CD66b, CD123, Glycophorin A) cells were removed by using the EasySep human monocyte enrichment kit without CD16 depletion .
- These lineage-negative CD14+ cells were cultured under 100 ng/ml human GM-CSF and 50 ng/ml human IL-4 in IMDM medium.
- Each cytokine was dissolved in PBS containing 0.1% BSA in 100 times the density used.
- For expansion, CD34+ cells were cultured under GM/SCF (100 ng/ml GM-CSF and 50 ng/ml SCF) or GM/IL-4 (100 ng/ml GM-CSF and 50 ng/ml IL-4) in IMDM medium.
- The medium was exchanged every 3 or 4 days.
- Cells were cultured in the MPC treatment plate (MD6 with Lid Low-Cell Binding; Nalge Nunc, ).
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Comparison of the phenotype and IL-12p70 production of mature HOCl-oxidized lysate-loaded DCs generated in human AB serum-free or serum-containing AIM-V and CellGenix DC media, and supplementation with different concentrations of GM-CSF and IL-4.
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500 IU/ml recombinant GM-CSF and 250 IU/ml recombinant IL-4 were sufficient concentrations for generating a favorable immature DC phenotype with low CD14, and intermediate HLA-DR and CD1c.
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2.3. Differentiation of hESC
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H1 hESCs were plated at 3 × 106 per well of 6-well ultralow attachment (ULA) plates in a total volume of 4 mL of medium'>X-VIVO-15 medium , supplemented with 1 mM sodium pyruvate, nonessential amino acids, 2 mM L-glutamine (all PAA Laboratories GmbH) and 5 μM 2-mercaptoethanol .
- The following growth factors were added: 50 ng/mL recombinant human bone morphogenetic protein-4 (BMP-4, & ), 50 ng/mL recombinant human vascular endothelial growth factor , 20 ng/mL recombinant human stem cell factor , and 50 ng/mL recombinant human granulocyte macrophage-colony stimulating factor (GM-CSF, & ).
- After 2-3 days, the medium was topped up with 2 mL of fresh supplemented X-VIVO-15 medium to produce a total volume of 6 mL.
- Subsequent feeding was performed every 2-3 days by replacing 2-3 mL of old medium with new supplemented X-VIVO-15 medium from which every 5 days a growth factor was removed starting with BMP-4 at day 5, followed by VEGF at…
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H1 hESCs were plated at 3 × 106 per well of 6-well ultralow attachment (ULA) plates in a total volume of 4 mL of medium'>X-VIVO-15 medium , supplemented with 1 mM sodium pyruvate, nonessential amino acids, 2 mM L-glutamine (all PAA Laboratories GmbH) and 5 μM 2-mercaptoethanol .
- The following growth factors were added: 50 ng/mL recombinant human bone morphogenetic protein-4 (BMP-4, & ), 50 ng/mL recombinant human vascular endothelial growth factor , 20 ng/mL recombinant human stem cell factor , and 50 ng/mL recombinant human granulocyte macrophage-colony stimulating factor (GM-CSF, & ).
- After 2-3 days, the medium was topped up with 2 mL of fresh supplemented X-VIVO-15 medium to produce a total volume of 6 mL.
- Subsequent feeding was performed every 2-3 days by replacing 2-3 mL of old medium with new supplemented X-VIVO-15 medium from which every 5 days a growth factor was removed starting with BMP-4 at day 5, followed by VEGF at day 10 and SCF at day 15 of differentiation [
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2.5. DC Maturation and Rapamycin Treatment
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Two days after monocytes were plated, monocyte-derived and hESC-derived immature DC were treated with 10 ng/mL and 5–7 ng/mL of rapamycin , respectively.
- On day 5, Cs were matured for 48 hr using a maturation cocktail consisting of 50 ng/mL of GM-CSF , 100 ng/mL IL-4 , 20 ng/mL IFNγ , 50 ng/mL TNFα , 10 ng/mL of IL-1β , and 1 μg/mL PGE2 .
- On day 6-7, DCs were harvested by gentle pipetting, passed through a 70 μm cell strainer, centrifuged, and resuspended prior to their use in experiments.
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Generation of imDC In Vitro
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Monocytes and CD3+ T cells were isolated from PBMCs by using anti-CD14 and anti-CD3 microbeads respectively [+ cells and CD3+ T cells was consistently ranging from 93% to 97%, as determined by flow cytometry.
- Monocytes were cultured in RPMI supplemented with 10% human AB serum, 10 ng/ml IL-4, and 50 ng/ml GM-CSF for 7 days to generate imDC as we described before [
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