Migration assays
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HUVECs were seeded at 15–20×103/well and cultured to 90% confluence on 24-well transwell tissue culture inserts (24-well plate with inserts, 5 µm pore/6.5 mm, TC-treated) as previously described 3 receptor agonists and antagonists were present during the migration in both transwell compartments.
- Migration experiments displayed an inter-donor variability in the absolute number of migrating T cells, thus for each experiment migration data have been normalized considering the number of counted cells in control conditions as 100%.
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Islet isolation, culture, and exposure to different beta-cell inhibitory substances
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Following the initial culture period, islets were cultured for an additional 6–24 hours in CMRL 1066 containing antibiotics, 2 mM glutamine and one of the following supplements: 0.5 mM sodium palmitate solubilized in 0.5% (weight/volume) fatty acid and lipopolysaccaride free bovine serum albumin (BSA) ; recombinant human Interleukin 1beta (IL-1ß (50 units/ml) and Interferon-gamma (IFN-γ) (1,000 units/ml) ; 100 mM hydrogen peroxide; 2 mM DETA/NO or 10 mM streptozotocin (STZ) .
- To some of the groups 10 µM of Imatinib was added at different time points prior to the addition of test substances given above.
- To controls equal amounts of vehicle (DMSO) were supplemented.
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The placenta releases sST2.
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C) Placental explants release sST2, secretion was increased by treatment with TNFα and IL-1β (100 ng/ml) and hypoxia-reperfusion injury, and was decreased under hypoxic conditions (n = 3).
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Effect of CORM-2 on HO-1 protein (A) and mRNA expression (B) in OA synoviocytes.
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Cells were stimulated with IL-1β (10 ng/ml) for 24 h and 16 h in the presence or absence of CORM-2 (50, 100, 200 µM) or RuCl3 (200 µM).
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Functional characterization of mutant N1.
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Cells were treated with IL-1β, lysed and the relative fold activation of NF-κB activity was determined.
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Infection of A549 cells for measurement of HBD2 gene expression and IL-8 levels
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The A549 cell line is a well characterized Type II alveolar epithelial tumor cell line which was obtained from the American Type Culture Collection .
- A549 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) with 10% Fetal Bovine Serum (FBS) and 1% Antibiotic-Antimycotic at 37°C in 5% CO2.
- All experiments were performed prior to the twenty fifth cell passage.
- A549 cells were plated at 1.5×105 cells/mL, in a volume of 3 mL of DMEM+10% FBS+1% Antibiotic-Antimycotic in 6-well tissue culture treated plates and incubated for 72 h at 37°C, in humidified 5% CO2.
- The cells were then washed 3 times with 3 mL Iscove's Modified Dulbecco's Medium (with sodium bicarbonate; without L glutamine:), and the media was replaced with 2 mL Iscoves+5% human serum ( AB Serum, ).
- Wells were infected with M.
- abscessus variants at…
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The A549 cell line is a well characterized Type II alveolar epithelial tumor cell line which was obtained from the American Type Culture Collection .
- A549 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) with 10% Fetal Bovine Serum (FBS) and 1% Antibiotic-Antimycotic at 37°C in 5% CO2.
- All experiments were performed prior to the twenty fifth cell passage.
- A549 cells were plated at 1.5×105 cells/mL, in a volume of 3 mL of DMEM+10% FBS+1% Antibiotic-Antimycotic in 6-well tissue culture treated plates and incubated for 72 h at 37°C, in humidified 5% CO2.
- The cells were then washed 3 times with 3 mL Iscove's Modified Dulbecco's Medium (with sodium bicarbonate; without L glutamine:), and the media was replaced with 2 mL Iscoves+5% human serum ( AB Serum, ).
- Wells were infected with M.
- abscessus variants at a concentration of 1×107 CFU/well.
- Uninfected control wells were left untreated or in some cases received either 100 ng/mL MALP-2 (TLR6/TLR2 ligand), or 20 ng/mL recombinant human interleukin-1β (IL1ß) .
- The plates were then incubated at 37°C, in humidified 5% CO2 for 8 hr.
- The wells were washed 3 times with 3 mL Iscove's medium, and 350 µLRLT buffer was added to the wells to lyse the cells.
- Lysates were placed in RNAse free tubes (VWR) and frozen −80°C for later RNA isolation.
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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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IL-12, IL-1β and TNF-α from BCG-, M. vaccae- and M. obuense-treated CD4+ cells activate Vδ2+ cells.
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b PBMCs were cultured overnight with mycobacteria in the presence of blocking antibodies to IL-12, IL-1β and TNF-α each at 100 μg/ml.
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Isolation of PBMCs and Monocyte-derived DC Generation
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The DC generation procedure was followed as previously described by Romani et al.
- and Kim et al.
- 6/well) were incubated for 5 days in medium'>AIM medium'>V medium containing 800 U/mL of animal-free (AF) recombinant interleukin 4 (rhIL-4) and 1000 U/mL AF-recombinant granulocyte-monocyte colony-stimulating factor (rhGM-CSF) to induce immature DC (iDC).
- Fresh medium containing these cytokines was replaced every 3 days.
- On day 5, to induce mature DCs (mDCs), iDCs were exposed to a cytokine cocktail containing AF-recombinant IL-1β (1000 U/mL) , IL-6 (1000 U/mL) and TNF-α (1000 U/mL) .
- After 6 h, a 10 µg/mL final concentration of PolyI:C was added to iDCs and cultured for 48 h. At day 7 or 8, the DC culture supernatant was collected and frozen at −80°C.
- Then, mDCs were harvested and stained with PE or FITC-conjugated antibodies…
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The DC generation procedure was followed as previously described by Romani et al.
- and Kim et al.
- 6/well) were incubated for 5 days in medium'>AIM medium'>V medium containing 800 U/mL of animal-free (AF) recombinant interleukin 4 (rhIL-4) and 1000 U/mL AF-recombinant granulocyte-monocyte colony-stimulating factor (rhGM-CSF) to induce immature DC (iDC).
- Fresh medium containing these cytokines was replaced every 3 days.
- On day 5, to induce mature DCs (mDCs), iDCs were exposed to a cytokine cocktail containing AF-recombinant IL-1β (1000 U/mL) , IL-6 (1000 U/mL) and TNF-α (1000 U/mL) .
- After 6 h, a 10 µg/mL final concentration of PolyI:C was added to iDCs and cultured for 48 h. At day 7 or 8, the DC culture supernatant was collected and frozen at −80°C.
- Then, mDCs were harvested and stained with PE or FITC-conjugated antibodies against CD11c, CD80, CD83, CD86, CD14, and HLA-DR and their appropriate isotype-matched regents .
- Cell surface expression was assayed by flow cytometry ( FACSCalibur, , ), and Cellquest was used to analyze the magnitude of surface molecule expression on the mDCs.
- IL-12 and IL-10 levels were determined by enzyme-linked immunosorbent assay (ELISA) according to manufacturer’s instructions.
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Generation of DC
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Blood samples from healthy donors are collected after informed consent, in accordance with the Declaration of Helsinki and approval of the of the University Hospital of the Ludwig-Maximilians-University, Munich, Germany.
- Peripheral blood mononuclear cells (PBMC) are isolated by Ficoll density gradient centrifugation.
- PBMC are resuspended in 15 ml VLE (very low endotoxin) medium'>RPMI 1640 medium supplemented with 1.5% human serum medium'>(DC medium) at 7.5′107 cells per 75 cm2 culture flask (NUNC, 178905) and incubated at 37°C and 5% CO2 for 1 h. Non-adherent cells are carefully removed by washing.
- Adherent monocytes are cultured in medium containing 100 ng/ml GM-CSF (Leukine® by Berlex, NC50419-050-30) and 20 ng/ml interleukin-4 (& 104-IL-050-CF) and fed with the same medium on days 3 and 6.
- On day 6 of culture, the immature C are differentiated into mC by addition of medium containing 10 ng/ml IL-1β…
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Blood samples from healthy donors are collected after informed consent, in accordance with the Declaration of Helsinki and approval of the of the University Hospital of the Ludwig-Maximilians-University, Munich, Germany.
- Peripheral blood mononuclear cells (PBMC) are isolated by Ficoll density gradient centrifugation.
- PBMC are resuspended in 15 ml VLE (very low endotoxin) medium'>RPMI 1640 medium supplemented with 1.5% human serum medium'>(DC medium) at 7.5′107 cells per 75 cm2 culture flask (NUNC, 178905) and incubated at 37°C and 5% CO2 for 1 h. Non-adherent cells are carefully removed by washing.
- Adherent monocytes are cultured in medium containing 100 ng/ml GM-CSF (Leukine® by Berlex, NC50419-050-30) and 20 ng/ml interleukin-4 (& 104-IL-050-CF) and fed with the same medium on days 3 and 6.
- On day 6 of culture, the immature C are differentiated into mC by addition of medium containing 10 ng/ml IL-1β (& 201-LB-025-CF), 15 ng/ml IL-6 (& 206-IL-050-CF), 10 ng/ml TNFα (& 210-TA-050-CF) and 1 µg/ml PGE2 for 2 d.
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