Cell culture
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To elucidate the effect of exogenous IL-11 on the regulation of versican isoforms, we performed treatment assays with different concentrations of rhIL-11.
- The gastric carcinoma cells were seeded at an equal density in cell culture flasks containing growth medium as described above.
- After 24 h, triplicate samples of subconfluent cells were treated with four different concentrations (0, 20, 50, 100 and 200 ng/ml) of activated rhIL-11 protein and incubated for 24 h. The cells were harvested to prepare mNA and protein as described below.
- In the time-point assays, the cells were treated with 100 ng/ml of rhIL-11 and harvested at three different time points: hours 24, 48 and 72.
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Cell lines and cell culture
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293T ( .
- number CRL-11268) and NIH3T3 ( .
- number CRL-1658) cells were both cultured in medium'>Dulbecco's medium'>modified medium'>Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS) and 1% Penicillin-Streptomycin with 5% CO2 at 37°C.
- Bone marrow cells (BM) isolated from wild type (wt) C57BL/6 mice were cultured in StemSpan Serum-free expansion medium supplemented with 1% glutamine, 1% penicillin-streptomycin, 100 ng/mL each murine stem cell factor , human Flt3-ligand and human interleukin 11 , and 10 ng/mL murine interleukin 3 .
- The KSHV-positive PEL cell line BCBL-1
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Cell Culture
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Human cord blood was collected following normal pregnancies and deliveries upon informed consent of the parents, in accordance with the ethical committee of the IRCCS Policlinico San Matteo Foundation and the principles of the Declaration of Helsinki.
- CD34+ cells from human cord blood samples were separated and cultured as previously described + progenitor cells were separated using immunomagnetic beads selection and cultured, for 13 days, in medium'>Stem medium'>Span medium (medium'>Stem cell; , ) supplemented with 1% L-glutamine, 100 U/ml penicillin, 100 ng/mL streptomicyn, 10 ng/mL IL-6, IL-11, 10 or 100 ng/mL rHuTPO ( EC , , ), and 100, 1000 or 2000 ng/mL AMG531 at 37°C in a 5% CO2 fully humified atmosphere.
- celltype'>MO7e cell line (Genetics Institute, Boston, MA) was cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 1% L-glutamine,…
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Human cord blood was collected following normal pregnancies and deliveries upon informed consent of the parents, in accordance with the ethical committee of the IRCCS Policlinico San Matteo Foundation and the principles of the Declaration of Helsinki.
- CD34+ cells from human cord blood samples were separated and cultured as previously described + progenitor cells were separated using immunomagnetic beads selection and cultured, for 13 days, in medium'>Stem medium'>Span medium (medium'>Stem cell; , ) supplemented with 1% L-glutamine, 100 U/ml penicillin, 100 ng/mL streptomicyn, 10 ng/mL IL-6, IL-11, 10 or 100 ng/mL rHuTPO ( EC , , ), and 100, 1000 or 2000 ng/mL AMG531 at 37°C in a 5% CO2 fully humified atmosphere.
- celltype'>MO7e cell line (Genetics Institute, Boston, MA) was cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 1% L-glutamine, 100 U/ml penicillin, 100 ng/mL streptomicyn, 10% Fetal Bovine Serum (FBS) and 50 ng/mL GM-CSF ( EC ).
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Myeloid Immortalization Assay
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We essentially followed the protocol developed by Modlich et al for the myeloid immortalization assay -) BM cells were isolated from 5 female C57BL6/J mice (6–8 weeks old ) using Lineage Cell Depletion Kit .
- Lin– BM cells were prestimulated for 2 days in’s Modified Dulbecco’s Medium containing 50 ng/ml recombinant murine stem cell factor, 100 ng/ml recombinant human Flt-3 ligand, 100 ng/ml recombinant human IL-11, 10 ng/ml recombinant murine IL-3 , 10% fetal calf serum, 1% penicillin/streptomycin, and 2 mmol/l glutamine (here after refer as I10-S3F11 medium) at a density of 5×105 cells/ml.
- The cells were transduced on days 3, 4, 5, and 6 using 105 cells and an MOI of 10–30 per transduction.
- For each vector or vector dose, three replicate transductions were performed in three different wells of a 24 well tissue culture plate and vector copy number for each…
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We essentially followed the protocol developed by Modlich et al for the myeloid immortalization assay -) BM cells were isolated from 5 female C57BL6/J mice (6–8 weeks old ) using Lineage Cell Depletion Kit .
- Lin– BM cells were prestimulated for 2 days in’s Modified Dulbecco’s Medium containing 50 ng/ml recombinant murine stem cell factor, 100 ng/ml recombinant human Flt-3 ligand, 100 ng/ml recombinant human IL-11, 10 ng/ml recombinant murine IL-3 , 10% fetal calf serum, 1% penicillin/streptomycin, and 2 mmol/l glutamine (here after refer as I10-S3F11 medium) at a density of 5×105 cells/ml.
- The cells were transduced on days 3, 4, 5, and 6 using 105 cells and an MOI of 10–30 per transduction.
- For each vector or vector dose, three replicate transductions were performed in three different wells of a 24 well tissue culture plate and vector copy number for each well is separately measured later on.
- Virus preloading was carried out on RetroNectin-coated suspension culture dishes by spinoculation for 30 minutes at 4°C.
- The transduction starts with 1×105 cells seeded into 500 µl medium'>medium and was increased by 250 µl medium'>medium on the following days, so that the culture volume was 1.25 ml on day 6.
- On day 10 (four days after the final addition of virus), an aliquot of cells were taken and treated with Benzonase Nuclease at a concentration of 50 units/ml for one hour at 37 degree.
- Genomic DNA was then extracted from the cells for vector copy number measurement using quantitative PCR.
- After retroviral transduction, the BM cells were expanded as mass cultures for 2 weeks in the I10-S3F11 medium.
- Cell concentration was maintained below 2×106/ml by changing medium every 2–3 days.
- After mass culture expansion for 14 days the BM cells from each transduction were plated into a single 96-well plate at a density of 100 cells/well.
- Cells from the SFFV-GFP vector group at the highest dose were also plated at 10 cells/well into a 96-well plate.
- Two weeks later the wells with active cell growth were counted.
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