Macaque DCs were generated from bone marrow precursors as described earlier.32 (link) Briefly, after red blood cell lysis, macaque cells (1 × 106cells/ml) were cultured in complete medium supplemented with 1% macaque serum (collected from our animals) and recombinant human GM-CSF (100 U/ml, Novartis, Basel, Switzerland). On day 3, the supernatant was replaced by fresh medium containing GM-CSF, macaque serum and with or without recombinant human IL-10 (20 ng/ml, R&D Systems, France). On day 7, the adherent cells were harvested. In some cases, the adherent iBMDC and iBMDC10 were cultured overnight with purified recombinant rtTA protein (10 µg/ml) with or without recombinant human IL-10 (20 ng/ml, R&D Systems). Three pilot animals received autologous iBMDC10 ID in the inguinal lymph nodes area. For Mac 4 and Mac 5, autologous iBMDC10 were injected ID in the inguinal and popliteal lymph nodes areas of the rAAV-1 vector injected leg. Mac 6 to 10 received autologous iBMDC10 by IV route. Each animal received the first injection the day before the AAVr injection and the second one, 2 months later, during the first Dox induction of the cmEpo system. Mac 4 and Mac 5 received a third injection of iBMDC10 three months post-AAVr injection. Cell dose was ~1 × 106 iBMDC10/kg.
Recombinant human il 10
Manufactured by R&D Systems
Sourced in France, United States, Canada
Recombinant human IL-10 is a cytokine produced in E. coli. It has a molecular weight of approximately 18 kDa.
Automatically generated - may contain errors
Lab products found in correlation
Ns 398, by Cayman Chemical (1 mentions)
Anti il 10 monoclonal antibody, by R&D Systems (1 mentions)
Nor noha, by Cayman Chemical (1 mentions)
Recombinant mouse il 10, by R&D Systems (1 mentions)
Dulbecco phosphate buffered saline (dpbs), by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Mtt assay kit, by Cayman Chemical (1 mentions)
Gm csf, by R&D Systems (1 mentions)
Recombinant human gm csf, by Novartis (1 mentions)
Anti cd11c, by BD (1 mentions)
Interleukin 4 (il 4), by Merck Group (1 mentions)
Fluorescein isothiocyanate conjugated anti cd16, by BD (1 mentions)
Rapamycin, by Selleck Chemicals (1 mentions)
αpd l1 antibody, by BioXCell (1 mentions)
Tgf β, by R&D Systems (1 mentions)
Nivolumab, by Bristol-Myers Squibb (1 mentions)
Human peripheral blood monocytes, by STEMCELL (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Facsverse, by BD (1 mentions)
11 protocols using recombinant human il 10
1
Generating Macaque Dendritic Cells
2
Immunomodulatory Factors of iPSC-MSCs
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To evaluate the soluble factors produced by iPSC-MSCs, the monocytes were removed from the plates of co-culture (on day 5) and then iPSC-MSCs were cultured further in a fresh medium for an additional 24 h. Prostaglandin (PG)E2, IL-10, IL-6, and tumor necrosis factor-stimulated gene 6 (TSG-6) levels were determined in the supernatants of iPSC-MSCs cultured with or without monocytes. To evaluate the cytokines produced by DCs, DCs were collected from the co-cultures on day 7 and then cultured in the new plates for an additional 12 h. IL-10 or IL-12p70 levels were determined in the supernatants of DCs cultured with or without iPSC-MSCs. The factor levels were measured using an enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN, USA).
To investigate the role of soluble factors on the immunomodulatory effects of iPSC-MSCs, the following reagents were used for the co-culture systems: neutralizing anti-IL-6 (0.25 μg/mL; R&D Systems Europe, Abingdon, UK), anti-IL-10 monoclonal antibody (0.075 μg/mL; R&D Systems Europe), human recombinant IL-10 (0.5μg/mL; R&D Systems Europe), or NS-398 (5 μM; Cayman Chemical, Ann Arbor, MI, USA), an inhibitor of PGE2 synthesis.
To investigate the role of soluble factors on the immunomodulatory effects of iPSC-MSCs, the following reagents were used for the co-culture systems: neutralizing anti-IL-6 (0.25 μg/mL; R&D Systems Europe, Abingdon, UK), anti-IL-10 monoclonal antibody (0.075 μg/mL; R&D Systems Europe), human recombinant IL-10 (0.5μg/mL; R&D Systems Europe), or NS-398 (5 μM; Cayman Chemical, Ann Arbor, MI, USA), an inhibitor of PGE2 synthesis.
3
Stimulation of Cells with IL-10 and LPS
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Fresh media was added to the cells before stimulation experiments. Recombinant mouse IL-10 (R&D Systems) from a 1 mg/ml stock was diluted to a 20 μg/ml working stock in 0.1% BSA solution. It was then added to cells at a final concentration of 20 ng/ml (and 100 ng/ml for metabolic studies). LPS (Sigma E.coli O111:B4) was diluted from stock concentration of 1 mg/ml in complete DMEM, and used at a final concentration of 100 ng/ml. For LPS + IL-10 treatments, cells were pre-incubated for five minutes with IL-10 prior to the addition of LPS. Cells were typically stimulated for 24 hours before conducting further assays. Inhibitor experiments involved pre-treatment before LPS/IL-10 addition. Cells requiring incubation with inhibitor nor-NOHA (150 μM) (Cayman Chemicals) were pre-treated 1 h prior to stimulation. Dimethyl Sulfoxide (DMSO) and/or DPBS (Sigma) were used as controls for inhibitor experiments. For THP-1 cells, 100 ng/ml human recombinant IL-10 (R&D Systems) was used.
4
IL-10 Stimulation Effects on Cell Proliferation
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Cells were treated with recombinant human IL-10 (R & D Systems) at various doses (50, 100, or 500 U/ml) and for different times (at 6-hr intervals during a 72-hr culture period) accordingly to previously reported conditions [7 (link)]. Proliferation was measured using the MTT Assay Kit (Cayman Chemical Company, Michigan, USA).
5
Monocyte Differentiation Induction
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Recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 were purchased from Millipore (Bedford, MA, USA). Recombinant human IL-10 was purchased from R&D Systems (Minneapolis, MN, USA). Phenylarsine oxide (PAO), CD45 PTPase inhibitor and PTPase inhibitor XVIII were purchased from Merck Millipore (Bedford, MA, USA). Fluorescein isothiocyanate-conjugated anti-CD16, phycoerythrin-conjugated anti-CD163, anti-CD11c, -CD80, -CD1a and -CD14, and APC-conjugated anti-CD14 monoclonal antibodies (mAbs) were purchased from BD Pharmingen (San Diego, CA, USA).
6
Immune Modulator Reagent Preparation
7
ADCP Evaluation of Biosimilar Macrophage
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Human peripheral blood monocytes (STEMCELL Technologies, Vancouver, Canada) were differentiated into M2c macrophage using RPMI 1640 (Gibco) containing 10% FBS (Hyclone), 50 ng/mL of recombinant human macrophage colony-stimulating factor (M-CSF; R&D Systems), and 50 ng/mL of recombinant human IL-10 (R&D Systems). Using a CellTrace™ CFSE Cell Proliferation Kit, M2c macrophage was stained with fluorescent dye. For comparison, six lots each of SB3 and reference product (final concentration: 69 ng/mL) were administered to SKBR3 stained with CellTrace™ Far Red Cell Proliferation Kit. Following incubation, ADCP was induced for 4 h and analyzed with FACSVerse™ (BD Bioscience). ADCP activity was determined by flow cytometry. Target SKBR3 cells were labeled with Far Red dye and monocyte-derived M2c macrophages were labeled with CFSE dye. Phagocytosis was evaluated using a FACSVerse™ flow cytometer, and the percentage phagocytosis was calculated by dividing the number of double-positive cells by the total number of macrophage cells.
8
VEGF-Induced Capillary Formation in HUVECs
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The effects of IL-10 on the formation of capillary-like structures induced by vascular endothelial growth factor (VEGF) were investigated in the HUVECs grown on Matrigel per published protocols [24 (link)]. Cells were seeded at 1 × 103 cells/well on 96-well plates in wells coated with Cultrex Basement Membrane Extract (BME, R&D Systems). These were then cultured in serum-free M199 medium in the presence of vehicle (control), VEGF (50 ng/ml; recombinant human VEGF; R&D Systems) alone, VEGF (50 ng/ml) plus low-dose IL-10 (10 pg/ml; recombinant human IL-10; R&D Systems), VEGF (50 ng/ml) plus high-dose IL-10 (100 pg/ml), or high-dose IL-10 (100 pg/ml) alone. After incubation for 5 h, the total number of capillaries and tube-like structures (complete loop structures) per well in each treatment group were counted, and representative photos of the capillary-like structures were taken.
9
Immunological and Genetic Investigation of Primary Immunodeficiencies
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In addition to immunoglobulin levels and lymphocyte subpopulations (i.e., CD3+, CD4+, CD4 + CD45RA+, CD4 + CD45RO+, CD19+, CD19 + CD27+, and CD19 + CD27−), characteristics and well-syndromes of PIDs accompanying the SD phenotype were identified by experienced physicians, and were consistent with defective lymphocyte proliferation to mitogens and antigens, and phorbol myristate acetate-stimulated polymorphonuclear reactive oxygen species as represented by H2O2 production as previously described54 (link).
To evaluate IL-10 signalling in the patients with RD and unrecognized molecular defects at that time, purified PBMCs (5 × 106/ml) were stimulated overnight with 50 ng/ml E. coli LPS (Sigma-Aldrich, St. Louis, Mo) alone or with 20 ng/ml recombinant human IL-10 (R&D Systems, Minneapolis, MN). The supernatants were analysed using a commercially available TNF-α ELISA development kit in duplicate using a Tecan Sunrise ELISA micro-plate reader (R&D Systems, Minneapolis, MN). Candidate genetic analysis in the patients with the RD phenotype predisposing to IBD included, at least, IL10, IL10RA, IL10RB, NEMO, FOXP3, XIAP, STAT3 and STAT155 (link), 56 (link). Every two oligonucleotide primers were selected to cover the entire coding region by Sanger sequencing57 .
To evaluate IL-10 signalling in the patients with RD and unrecognized molecular defects at that time, purified PBMCs (5 × 106/ml) were stimulated overnight with 50 ng/ml E. coli LPS (Sigma-Aldrich, St. Louis, Mo) alone or with 20 ng/ml recombinant human IL-10 (R&D Systems, Minneapolis, MN). The supernatants were analysed using a commercially available TNF-α ELISA development kit in duplicate using a Tecan Sunrise ELISA micro-plate reader (R&D Systems, Minneapolis, MN). Candidate genetic analysis in the patients with the RD phenotype predisposing to IBD included, at least, IL10, IL10RA, IL10RB, NEMO, FOXP3, XIAP, STAT3 and STAT155 (link), 56 (link). Every two oligonucleotide primers were selected to cover the entire coding region by Sanger sequencing57 .
10
MCL Cell Proliferation Assay with Wnt5a and Cirmtuzumab
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MCL cells were labeled by carboxyfluorescein succinimidyl ester (CFSE, Life Technologies) and plated at 1.5 × 106/well/ml in a 24-well tray on a layer of irradiated HeLaCD154 cells (8000 Rad; 80 Gray) at a MCL/HeLaCD154 cell ratio of 15:1 in complete RPMI-1640 medium supplemented 5 ng/mL of recombinant human interleukin (IL)-4 (R&D Systems) and 15 ng/mL recombinant human IL-10 (R&D Systems). Wnt5a (200 ng/ml, R&D Systems) or cirmtuzumab (10 μg/ml) as indicated in the text. CFSE-labeled MCL cells were analyzed by flow cytometry; Modfit LT software (version 3.0, Verity Software House) was used for analysis of cell proliferation as previously described44,45.
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