Increased TfR expression in CaCo-2 cells is not due to enhanced proliferation.
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Flow cytometry analysis of CaCo-2 cells double stained for CFSE dilution assay and TfR (mouse anti-human TfR stain), after incubation with the following inflammation mediators: TNFα, IL1β, IL-6, a mixture of the three, or CRM from mouse source.
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Cells and cultures
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The human KG1 leukemic cell line, RPMI8226 myeloma (MM) cell line, HepG2 hepatoma cell line, DU145 prostate carcinoma cell line, and A-MB231 breast cancer cell lines were obtained from American Type Culture Collection (ATCC) .
- The MM cell line INA6 was kindly provided by Dr. Renate Burger (University of Kiel, Kiel, Germany).
- Bone marrow mononuclear cells (BMMCs) were isolated from fresh bone marrow aspirates of patients with myeloma and primary CD138+ myeloma cells were further sorted using CD138 MicroBeads as described previously
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Processing whole blood samples
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Peripheral (PB.1 and PB.2) and cord (CB.1 and CB.2) blood-derived CD34+ cells were obtained from AllCells .
- Blood collections were performed at AllCells and using standard, 8 ml Vacutainer Cell Processing Tubes (both sodium citrate and sodium heparin-based tubes are acceptable ; , ).
- Appropriate documentation for informed consent was completed prior to blood collection .
- Vacutainers were processed within 24 hours of collection.
- Briefly, the PBMC-containing upper phase was collected and washed with ice-cold PBS .
- Cells were either frozen down or used directly for purification with the CD34 MicroBead Kit and used according to the manufacturer's protocol.
- Some samples were treated with Histopaque ( ; St. Louis, ) to minimize the number of red blood cells and centrifuged at 2000 rpm for 20 minutes without braking.
- The interface containing the PBMCs was removed if samples were treated with histopaque, cells washed again with chilled…
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Peripheral (PB.1 and PB.2) and cord (CB.1 and CB.2) blood-derived CD34+ cells were obtained from AllCells .
- Blood collections were performed at AllCells and using standard, 8 ml Vacutainer Cell Processing Tubes (both sodium citrate and sodium heparin-based tubes are acceptable ; , ).
- Appropriate documentation for informed consent was completed prior to blood collection .
- Vacutainers were processed within 24 hours of collection.
- Briefly, the PBMC-containing upper phase was collected and washed with ice-cold PBS .
- Cells were either frozen down or used directly for purification with the CD34 MicroBead Kit and used according to the manufacturer's protocol.
- Some samples were treated with Histopaque ( ; St. Louis, ) to minimize the number of red blood cells and centrifuged at 2000 rpm for 20 minutes without braking.
- The interface containing the PBMCs was removed if samples were treated with histopaque, cells washed again with chilled PBS, centrifuged at 600× g for 15 minutes and either frozen down with CryoStor10 or used directly for purification.
- celltype'>CD34+ cell expansion media: StemSpan SFEM , , , each at a final concentration of 300 ng/ml, IL-6 (100 ng/ml) and IL-3 (10 ng/ml) , supplemented with DNaseI (final concentration at 20 U/ml), and 1× Antibiotic-antimycotic for overnight recovery.
- efined expansion media: serum-free StemSpan H3000 , animal-free IL-6 , and recombinant human IL-3, TPO, and SCF at the same concentrations listed above.
- PBMC expansion media: ExCyte Medium Supplement , Glutamax , SCF (250 ng/ml), IL-3 (20 ng/ml), Erythropoietin (2 U/ml; , ), IGF-1 (40 ng/ml), and Dexamethasone (1 µM; , ).
- PBMCs were resuspended at 1×106 cells/ml for expansion.
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Exposure of IRAK3-depleted THP-1 cells to additional stress results in more inflammation and ROS.
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Gene expression was analyzed using qRT-PCR and mROS production was determined by flow cytometry in THP-1 cells exposed to 5.5 mM D-glucose and 9.5 mM D-mannitol (osmotic control) or 15 mM D-glucose (n = 6), and 100 ng/ml IL-6 (n = 6).
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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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Cell Culture
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Frozen female CD34+ CBCs were supplied by Bio-Resource Center .
- C34+ CBCs were cultured in hematopoietic culture medium [serum-free X-Vivo10 containing 50 ng/mL IL-6 , 50 ng/mL sIL-6 , 50 ng/mL SCF , ten ng/mL TPO , and 20 ng/mL Flt3/4 ligand ].
- Reprogrammed cells were cultured in feeder-less primate ES cell medium Repro FF (, .
- No. RCHEMD004), ReproFF2 (ReproCELL, cat No. RCHEMD006), mTeSR1 ( catalog number 05850) or E8 (16) supplemented with five ng/mL bFGF (total bFGF ten ng/mL) on Pronectin F-coated dishes.
- Passage of human iPSCs was previously described
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Cell cultures
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Cord blood-derived mast cells were derived in supplemented medium'>StemPro-34 SFM medium including 100 ng/mL recombinant human SCF (hSCF , , ) and 10 ng/mL human IL-6 as previously described
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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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Purification and Transduction of Mouse and Human Hematopoietic Progenitors
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Mouse Hematopoietic Progenitors (mLin−) were purified from BM mononuclear cells of C57BL6/6J mice byCell Depletion .
- The mouse cells were then prestimulated in StemSpam SFEM (serum-free expansion medium, StemCells Technologies, Canada) with 50ng/mL mouse Stem Cell Factor (mSCF) , 100ng/mL human Interleukin-11 (hIL-11), 100ng/mL human Flt-3 Ligand (hFlt-3L) and 10ng/mL human Interleukin-3 (hIL-3) (ocky , , ) for 4-6 hours.
- After which, mLin− cells were transduced with the different lentivirus.
- On the other hand, cord blood was collected from mothers attending the Royal Hospital, , after informed consent and via a protocol approved by the Local Research Ethics.
- Mononuclear cells (MNC) were obtained by Ficoll density centrifugation and ammonium chloride red cell lysis.
- Density-separated CB MNCs were depleted for lineage marker positive cells via theSep™ system ( Cell , ) according to the manufacturer’s instructions to generate Lineage negative (Lin−)…
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Mouse Hematopoietic Progenitors (mLin−) were purified from BM mononuclear cells of C57BL6/6J mice byCell Depletion .
- The mouse cells were then prestimulated in StemSpam SFEM (serum-free expansion medium, StemCells Technologies, Canada) with 50ng/mL mouse Stem Cell Factor (mSCF) , 100ng/mL human Interleukin-11 (hIL-11), 100ng/mL human Flt-3 Ligand (hFlt-3L) and 10ng/mL human Interleukin-3 (hIL-3) (ocky , , ) for 4-6 hours.
- After which, mLin− cells were transduced with the different lentivirus.
- On the other hand, cord blood was collected from mothers attending the Royal Hospital, , after informed consent and via a protocol approved by the Local Research Ethics.
- Mononuclear cells (MNC) were obtained by Ficoll density centrifugation and ammonium chloride red cell lysis.
- Density-separated CB MNCs were depleted for lineage marker positive cells via theSep™ system ( Cell , ) according to the manufacturer’s instructions to generate Lineage negative (Lin−) cells.
- Lin− cells were pre-stimulated in StemSpam SFEM and supplemented with 50ng/mL human Stem Cell Factor (hSCF), 50ng/mL human Flt-3 Ligand (hFlt-3L), 20ng/mL human Thrombopoietin (hTPO) and 10ng/mL human Interleukin-6 (hIL-6) for 4-6 hours.
- Lentiviral supernatants were added at a multiplicity of infection of 30 (for single transduction) or 20 (for each lentivirus during the double transduction).
- All the transductions were carried out over-night in the presence of 4mg/mL of.
- The efficiency of transduction was analyzed at four days by eGFP or / and mCherry expression.
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Liquid Cultures assays
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Two types of liquid culture were used one for maintaining stem/progenitors and the second for inducing myeloid differentiation.
- For the maintenance of HSC/progenitors, transduced Lin− cells were cultured in IMDM / 10% Fetal Calf Serum (FCS) supplemented with 20ng/mL SCF, 50ng/mL Interleukin-3 (IL-3), 20ng/mL IL-6 and 10ng/mL Granulocyte-Colony Stimulating Factor (G-CSF) .
- Fresh media was added every week.
- For Myeloid promoting differentiation, transduced cells were cultured as previously described for two weeks ( in IMDM/15% FCS supplemented with 2ng/ml IL-3 and 20ng/ml SCF.
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