The cell proliferation ability was determined using the Cell Counting Kit-8 (CCK8, Dojindo, Japan). Briefly, 3000 cells were seeded into 96-well plates (Corning, USA). At the indicated times, each well medium was replaced with an equal volume of fresh medium containing 10% CCK-8 reagent. After cells were incubated at 37 °C for 2.5 h, absorbance was measured using an Enzyme-linked immunosorbent instrument (Thermo-Fisher, USA) at 450 nm. Recombinant human interleukin 10 (rhIL-10, R&D Systems, USA) and its neutralizing antibody (Anti-IL-10, Abcam, UK) were used in this study.
Rhil 10
Manufactured by R&D Systems
Sourced in United States
RhIL-10 is a recombinant human interleukin-10 (IL-10) protein produced in a human cell expression system. IL-10 is an anti-inflammatory cytokine that plays a key role in regulating the immune response.
Automatically generated - may contain errors
Lab products found in correlation
Rhil 4, by R&D Systems (2 mentions)
Rhifn γ, by R&D Systems (2 mentions)
96 well plate, by Corning (1 mentions)
Cell counting kit 8 cck 8, by Dojindo Laboratories (1 mentions)
Enzyme linked immunosorbent instrument, by Thermo Fisher Scientific (1 mentions)
Anti il 10, by Abcam (1 mentions)
Rhil 2, by Thermo Fisher Scientific (1 mentions)
Rhil 7, by R&D Systems (1 mentions)
Rhil 15, by Thermo Fisher Scientific (1 mentions)
Rhil 12, by Thermo Fisher Scientific (1 mentions)
Rhil 6, by R&D Systems (1 mentions)
Rhil 21, by R&D Systems (1 mentions)
Ifn α, by R&D Systems (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Sepmate 50 tube, by STEMCELL (1 mentions)
Tryple select, by Thermo Fisher Scientific (1 mentions)
Versene, by Thermo Fisher Scientific (1 mentions)
Lymphoprep, by STEMCELL (1 mentions)
Tgf β1, by R&D Systems (1 mentions)
Rhtgf β1, by R&D Systems (1 mentions)
9 protocols using rhil 10
1
Cell Proliferation Assay with CCK8
2
Modulation of Human CD8+ T Cell Activation
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Purified naive and total human CD8+ T cells were cultured in RPMI 1640 medium supplemented with 10% FBS, 1% penicillin/streptomycin, and 1% L-glutamine, respectively. Cells were seeded at 5 to 10×104 cells into 96-well U bottom plate and stimulated with anti-CD3/CD28 coated microbeads (Dynabeads® T-Activator CD3/CD28; Thermo Fisher Scientific, Waltham, MA, USA) in the absence or presence of the indicated cytokines; recombinant human (rh) IL-1β (25 ng/ml), rhIL-6 (6.25 to 100 ng/ml), rhIL-21 (0.1 to 100 ng/ml), rhIL-7 (50 ng/ml), IFN-α (50 or 100 ng/ml), rhIL-10 (6.25 to 100 ng/ml; all from R&D systems, Minneapolis, MN, USA), rhIL-12 (50 ng/ml), rhIL-15 (50 or 100 ng/ml), rhIL-2 (100 IU/ml, all from PeproTech, Rocky Hill, NJ, USA). In some experiments, cells were stimulated with anti-CD3/CD28 coated microbeads with chemical inhibitors: STAT1 inhibitor Fludarabin, STAT3 inhibitor 5,15-DPP, or STAT5 inhibitor CAS 285986-31-4 (all from Sigma-aldrich, St. Louis, MO, USA) .
3
Isolation and Differentiation of Monocyte-Derived Macrophages
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Peripheral blood mononuclear cells (PBMCs) were isolated from Leukocyte Reduction System chambers (Bloodworks Northwest) with a Lymphoprep (Stemcell Technologies) density gradient applied to SepMate-50 tubes (Stemcell Technologies) and cryopreserved in FBS/10% dimethylsulfoxide (DMSO; Sigma). CD14+ monocyte isolation from cryopreserved PBMCs was performed using EasySep Human CD14 Positive Selection Kits (Stemcell Technologies), according to the manufacturer’s instructions. With minor variations in timing, macrophages were generated by culturing CD14+ monocytes in RP10 with 10 ng/mL recombinant human (rh) granulocyte-macrophage colony-stimulating factor (GM-CSF; R&D Systems) for 6 days at 37°C, 5% CO2, refreshing cytokine on day 3. Macrophages were detached using 0.25% Trypsin-EDTA (Gibco) and cell scrapers, and replated in RP10. For transduction, lentivirus was added to culture and macrophages harvested for downstream assays 5–7 days later, replacing media every 2–4 days. For downstream assays, macrophages were detached using 0.25% Trypsin-EDTA, TrypLE, TrypLE Select or Versene (Gibco) and cell scrapers. To test IL-12 receptor (IL-12R) expression, macrophages were cultured in 100 ng/mL lipopolysaccharide (LPS) (Sigma) and 20 ng/mL each rhIFNγ, rhIL-4, rhIL-10 and rh transforming growth factor beta 1 (TGF-β1) (all R&D).
4
Cytokine Modulation of THP-1 Macrophages
5
Intracellular Cytokine Staining Assay
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Stock solution (10 µg/ml) of rhIL-10 (R&D Systems, Abingdon, UK) was
reconstituted in Tris-NaCl buffer (TBS; Sigma, St Louis, MO, USA). Brefeldin A
(10 mg/ml) was purchased from Sigma Aldrich (Oslo, Norway). The monoclonal
antibodies (abs) anti-CD45 fluorescein isothiocyanate (FITC) (clone J33),
anti-CD3-phycoerythrin-cyanine 7 (PE-Cy7) (clone UCHT1),
anti-CD14-allophycocyanin (APC) (clone RMO52), anti-TNF-α phycoerythrin (PE)
(clone IPM2) and the PerFix-nc kit used for intracellular staining were
purchased from Beckman Coulter (Indianapolis, IN, USA). Anti-IL-6 PE abs (clone
MQ2-13A5) and anti-IL-8 PE abs (clone B-K8) were purchased from Nordic Biosite
(Oslo, Norway).
reconstituted in Tris-NaCl buffer (TBS; Sigma, St Louis, MO, USA). Brefeldin A
(10 mg/ml) was purchased from Sigma Aldrich (Oslo, Norway). The monoclonal
antibodies (abs) anti-CD45 fluorescein isothiocyanate (FITC) (clone J33),
anti-CD3-phycoerythrin-cyanine 7 (PE-Cy7) (clone UCHT1),
anti-CD14-allophycocyanin (APC) (clone RMO52), anti-TNF-α phycoerythrin (PE)
(clone IPM2) and the PerFix-nc kit used for intracellular staining were
purchased from Beckman Coulter (Indianapolis, IN, USA). Anti-IL-6 PE abs (clone
MQ2-13A5) and anti-IL-8 PE abs (clone B-K8) were purchased from Nordic Biosite
(Oslo, Norway).
6
Modulation of IL-8 Expression in HT-29Cl16E Cells
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HT-29Cl16E cells (Ephyscience) were cultured in Dulbecco's modified Eagle's medium (4.5 g/liter glucose) supplemented with glutamax, 10% calf serum, 100 µg/ml streptomycin, and 100 U/ml penicillin at 37°C in a 5%-CO2 environment as previously described (38). Nox1 stealth RNAi siRNA or stealth RNAi siRNA negative control Med GC (Life Technologies) was transfected into cells using Lipofectamine ™ RNAimax reagent (Life Technologies). Cells were maintained in the same medium for 48 hours. Twenty-four hours before cell harvesting, human recombinant IL-10 (rhIL-10, 50 ng/ml, R&D System) was added to the medium. Cells were treated with TM (5 µg/ml), Tg (5 µM, Sigma-Aldrich) or DMSO 6 hours before harvesting. Cell supernatants were collected 48 hours after siRNA transfection. IL-8 expression was measured by ELISA using the BD OptEIA kit (BD Biosciences) according to the manufacturer’s instructions.
7
Cytokine Modulation of NK Cell Chemokine Receptors
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NK cells were freshly isolated from healthy donor PBMCs (n = 3 donors) using Rosette Sep (Stem Cell Technologies, Vancouver, BC, Canada). Fresh NK cells were resuspended in NK media without IL-2 at a concentration of 5 × 105 cells/mL in a 24-well cell culture plate (Corning). Next, 20 ng/mL rh (recombinant human) G-CSF (Amgen, Thousand Oaks, CA, USA), 200 U/mL IL-2, 50 ng/mL rhTNF-α (R&D Systems, Minneapolis, MN, USA), 200 ng/mL rhIFN-γ (R&D Systems), 20 ng/mL rhIL-4(R&D Systems), 20 ng/mL rhIL-15(R&D Systems), 20 ng/mL rhGM-CSF (R&D Systems), 20 ng/mL rhIL-10 (R&D Systems), 5 ng/mL rhTGF-β (R&D Systems), 50 ng/mL rhCCL5 (R&D Systems), 50 ng/mL rhCCL3 (R&D Systems), 50 ng/mL rhVEGF165 (R&D Systems), or 100 ng/mL rhSDF-1α (R&D Systems) were individually added to each well. NK cells in media with no cytokine were used as the control. All samples were incubated for 24 h in 37 °C 6.5% CO2. Cells from each sample were collected and stained for flow cytometry-based identification of CCR1, CCR5, CXCR4, and CXCR6 surface expression.
8
IL-10 Modulates PBMC Immune Responses
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Cryopreserved PBMC (5 HUU birth and 5 HUU 6-month samples) were thawed and counted as described above. Cells were stimulated for 24h in complete RPMI+10% FBS with TLR cocktail alone or in combination with rhIL-10 (20ng/mL, R&D Systems) or anti-hIL-10 (500ng/mL, R&D Systems). BFA (5ug/mL) was added for the last 4 hours of culture. Cells were washed with PBS and stained with Zombie Yellow Fixable Viability, followed by surface staining with HLA-DR Alexa Fluor 488, CD19 PerCP-Cy5.5, CD16 APC, CD56 APC-Cy7 (Biolegend), Gamma delta TCR PE and CD3 Alexa Fluor 700 (BD Biosciences). Cells were washed and treated with eBioscience Foxp3/Transcription Factor Staining Buffer Set (ThermoFisher) then stained for IL-10 PE-Dazzle 594, IFNγ BV421 (Biolegend) and FoxP3 PE-Cy7 (eBiosciences). Cells were washed and fixed with PBS+1% paraformaldehyde before acquisition on the cytometer (Beckman Coulter Gallios).
9
Monocyte-T Cell Crosstalk Regulates IL-1β Release
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Monocytes or macrophages were plated at 40,000 cells in a 96-well flat-bottom plate with 200,000 expanded T cells (T cells were 4 days postactivation at the time of assay) and cocultured for 16 hours. Myeloid cells were stimulated with 10 ng/mL lipopolysaccharide for 5 hours with 5 mmol/L adenosine triphosphate added for the last hour (both from Cedarlane Laboratories, Burlington, Ontario, Canada). The caspase 1 inhibitor Y-VAD (Cedarlane Laboratories, 10 mg/mL) was added for the final 1.5 hours. Recombinant human (rh) IL10 (BD Biosciences), IL22 (BioLegend), IL17A, and IFNG (both from eBioscience) were added as indicated. Unless indicated otherwise, rhIL10 was added at 5 ng/mL. After 5 hours, supernatants were collected, and IL1B was measured using enzyme-linked immunosorbent assay (R&D Systems). Results from experiments with monocytes or monocyte-derived macrophages were equivalent (data not shown).
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