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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Tumor-specific IFN-γ release and cell-mediated cytotoxicity after vaccination with mGC8 cells and GM-CSF.
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Vaccination with mGC8 with or without LP, GM-CSF, and IFA.
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Tumor cell vaccination (prophylactic/therapeutic), LRAST
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To determine the immunogenicity of the tumor cells, 107 tumor cells were irradiated with 10,000 rad and subcutaneously injected into mice.
- Two weeks later, the mice were challenged by subcutaneous injection of 3 × 106 viable tumor cells into the opposite flank.
- Experimental groups generally consisted of 5 mice.
- Tumor development was followed by serial measurements of the tumor diameter and is depicted as tumor size (mm2) = d × D, where d and D were the shortest and the longest tumor diameter, respectively.
- Animals were euthanized when D reached 10 mm.
- Lymphopenia was induced by i.p.
- injection of cyclophosphamide (200 mg/kg, , ).
- This dose was chosen since earlier studies have shown an increased proliferation and long-term survival of antigen-specific T cells at this dose of cyclophosphamide, alone or in combination with fludarabine [7 naïve syngeneic splenocytes followed by s.c. vaccination with irradiated mGC8 cells (107, 10,000 rad) with or without a…
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To determine the immunogenicity of the tumor cells, 107 tumor cells were irradiated with 10,000 rad and subcutaneously injected into mice.
- Two weeks later, the mice were challenged by subcutaneous injection of 3 × 106 viable tumor cells into the opposite flank.
- Experimental groups generally consisted of 5 mice.
- Tumor development was followed by serial measurements of the tumor diameter and is depicted as tumor size (mm2) = d × D, where d and D were the shortest and the longest tumor diameter, respectively.
- Animals were euthanized when D reached 10 mm.
- Lymphopenia was induced by i.p.
- injection of cyclophosphamide (200 mg/kg, , ).
- This dose was chosen since earlier studies have shown an increased proliferation and long-term survival of antigen-specific T cells at this dose of cyclophosphamide, alone or in combination with fludarabine [7 naïve syngeneic splenocytes followed by s.c. vaccination with irradiated mGC8 cells (107, 10,000 rad) with or without a s.c. injection of GM-CSF (1 μg, , ) diluted in HBSS and emulsified with an equal volume of incomplete Freund's adjuvant (IFA, Taufkirchen, ) as described elsewhere [6) were injected 4 days before vaccination and tumor vaccinations were repeated every two weeks for a total of 4 vaccinations.
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BMDC culture
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Bone marrow was extracted from the femur and tibia passed through a screen and washed in cold media.
- 2×106 bone marrow cells were resuspended in 10 ml of DC media (RPMI, 10% LPS free FCS, β-mercaptoethanol, L-glutamine, 40 ng/ml GM-CSF and cultured in 10 cm petri dishes at 37°C.
- On day 3, 10 ml of DC media was added to the culture.
- On day 6 and 8, 10 ml of culture was removed, resuspended in 10 ml of fresh DC media and added back to the culture.
- On day 9, BMDCs were resuspended in DC media− (ex GM-CSF) at 2×106cell/ml, re-plated in 24 well plates and stimulated with LPS (100 ng/ml), Poly I∶C (100 µg/ml), and/or IFNα (1000 u/ml).
- On day 10, BMDCs were pulsed for 2 hours with gp33 peptide alone or gp33, gp276 and gp61 peptides (10−6 M) respectively, and washed prior to use in in…
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Bone marrow was extracted from the femur and tibia passed through a screen and washed in cold media.
- 2×106 bone marrow cells were resuspended in 10 ml of DC media (RPMI, 10% LPS free FCS, β-mercaptoethanol, L-glutamine, 40 ng/ml GM-CSF and cultured in 10 cm petri dishes at 37°C.
- On day 3, 10 ml of DC media was added to the culture.
- On day 6 and 8, 10 ml of culture was removed, resuspended in 10 ml of fresh DC media and added back to the culture.
- On day 9, BMDCs were resuspended in DC media− (ex GM-CSF) at 2×106cell/ml, re-plated in 24 well plates and stimulated with LPS (100 ng/ml), Poly I∶C (100 µg/ml), and/or IFNα (1000 u/ml).
- On day 10, BMDCs were pulsed for 2 hours with gp33 peptide alone or gp33, gp276 and gp61 peptides (10−6 M) respectively, and washed prior to use in in vitro proliferation assays or intravenous infusions into treated mice.
- For intravenous infusions, 2×106 BMDCs prepared in 200 µl of sterile HBSS were given to each treated mouse.
- For in vivo treatment, mice were infused with 10000 u of IFNα.
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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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Generation of BMDC and BMMC
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Complete medium'>medium'>RPMI (cmedium'>medium'>RPMI), medium'>medium'>RPMI 1640 medium containing 10% fetal calf serum , was used as culture medium.
- For BMDC induction, 5×106 BM cells were cultured supplemented with 10 ng/ml recombinant murine GM-CSF for five days
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GC frass-induced PAR-2 mRNA and cytokine production from BMDC.
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Bone marrow was isolated from BALB/c mice and cultured in the presence of GM-CSF for 6 days.
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GM-CSF induces CD103 expression on Flt3L-derived CD8α-equivalent DCs.
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FACS analysis of developing DCs in bone marrow cells from C57BL/6, Batf3
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Phagocytic cells assays
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The monocyte/macrophage J774.3 cell line used in this study was kindly provided by Dr. María Inés Becker .
- J774.3 cells were routinely grown in high-glucose medium'>DMEM medium (GIBCO), supplemented with 10% Fetal Bovine Serum and 1 mM HEPES (GIBCO) in T75 bottles.
- Cells were incubated at 37°C and 5% CO2 until 95% of confluence.
- Before infection assays, cells were treated with 0.1 mg/ml trypsine for 5 min, recovered in 50 ml polypropylene tubes, and centrifuged at 1,800× g for 5 min at room temperature.
- After three washes with supplemented DMEM medium, cell number and viability was determined in a haemocytometer, using the trypan blue staining (1 mg/ml).
- 5×105 cells/ml were seeded in 24 well-plates and incubated overnight at 37°C and 5% CO2.
- DCs were prepared from bone marrow precursors of C57BL/6 mice.
- Cells were incubated in complete RPMI 1640 medium supplemented with 5% FCS…
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The monocyte/macrophage J774.3 cell line used in this study was kindly provided by Dr. María Inés Becker .
- J774.3 cells were routinely grown in high-glucose medium'>DMEM medium (GIBCO), supplemented with 10% Fetal Bovine Serum and 1 mM HEPES (GIBCO) in T75 bottles.
- Cells were incubated at 37°C and 5% CO2 until 95% of confluence.
- Before infection assays, cells were treated with 0.1 mg/ml trypsine for 5 min, recovered in 50 ml polypropylene tubes, and centrifuged at 1,800× g for 5 min at room temperature.
- After three washes with supplemented DMEM medium, cell number and viability was determined in a haemocytometer, using the trypan blue staining (1 mg/ml).
- 5×105 cells/ml were seeded in 24 well-plates and incubated overnight at 37°C and 5% CO2.
- DCs were prepared from bone marrow precursors of C57BL/6 mice.
- Cells were incubated in complete RPMI 1640 medium supplemented with 5% FCS , 2 mM glutamine, 1 mM non-essential amino acids, 1 mM pyruvate, 1 mM HEPES, and 10 ng/ml of recombinant murine GM-CSF .
- All cell culture media were acquired from GIBCO .
- Culture media was replaced every 2 days.
- After 6 days, the phenotype of DCs was analyzed by flow cytometry for the expression of the surface markers CD11c, CD86 and CD40, which revealed over 70% CD11c+ with an immature phenotype.
- Before the infection assays, DCs were washed three times with PBS and then culture media was replaced with complete medium'>RPMI medium without antibiotics.
- DCs and macrophages were infected with S. Enteritidis PT1 at a multiplicity of infection (MOI) equal to 25.
- The MOI was confirmed by plating serial dilutions of bacterial cultures on LB agar.
- After 1 h of incubation at 37°C and 5% CO2, the supernatant of infected cells was recovered and stored for genomic DNA preparation.
- Cells were washed two times with PBS and 1 ml of appropriate medium supplemented with gentamicin 50 mg/ml was added to cell cultures to kill the remaining extracellular bacteria.
- After 2, 18 and 24 h of infection, cells were removed from the wells and centrifuged at 783× g for 5 min.
- No significant changes on cell viability were observed after infection with S. Enteritidis at the time points used on the experiments (data not shown).
- To recover intracellular bacteria, phagocytic cells were treated with 1 ml of lysis solution (19% ethanol, 0.1% SDS, 1% saturated basic phenol) for 30 min on ice.
- After the incubation period, the cell lysate was centrifuged at 7,043× g for 5 min, and genomic DNA was extracted following the methodology mentioned above.
- In order to quantify the amount of intracellular bacteria at different time points, either 100,000 DCs or 10,000 J774.3 cells were treated with 1 ml of PBS-0.1% triton X-100 for 15 min at room temperature.
- 100 µl of cell lysates were plated on LB agar and the plates were incubated at 37°C for 18 h. In parallel, an equal amount of bacteria used to infect cells was incubated in 1 ml of cell medium.
- The incubation was performed for 2 h and after that time bacteria were recovered by centrifugation at 7.043× g for 5 min, and genomic DNA was prepared as described above.
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Generation of bone marrow-derived DCs
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Mouse BMDCs were generated from bone marrow progenitor cells, according to the modified protocol of[
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