B cells stimulated with LPS (5 μg/ml) for 24 h were then washed with PBS to remove LPS. These B cells were then cotransferred with CD8+CD103− med, CD8+CD103+ iTreg, or nTreg cells (the ratio of T:B was 1:2, 8 million B cells per mouse) into B6 Rag1−/− mice (6 weeks age). In addition, TβRI inhibitor (ALK5) (1 mg/kg, Sigma-Aldrich), anti-IL-10R (1 mg/kg, Biolegend), or DMSO (control for ALK5i) or cIgG (control for anti-IL-10R) were given to some groups mice by i.p. injection at days 0 and 2. The mice were sacrificed as indicated time points and splenic B cells were harvested and used to measure Ki-67 expression in cells gated on B220+ by flow cytometry. Then we carried out another in vivo suppression assay. The established cGVHD lupus nephritis mice with evident lupus nephritis were depleted of endogenous CD3+ T cells (26 (link)) with a single-dose 300 mg anti-mouse CD3 Ab (ExCell Bio, Shanghai, CHN), or with the isotype control, and endogenous B cells were stimulated with a single 10 μg dose of LPS (Sigma-Aldrich). Three days later, CD8+CD103− med, CD8+CD103+ iTregs, or nTregs were adoptively transferred to the mice with anti-CD3 Ab treatment. The percentages of plasma cells (CD138+) were detected and sera were collected 0, 1, and 2 weeks after cell transfer for IgG measurement.
Anti il 10r
Manufactured by BioLegend
Anti-IL-10R is a laboratory reagent used for research purposes. It functions as an antibody that binds to and blocks the interleukin-10 receptor (IL-10R), a cell surface protein involved in regulating immune responses.
Automatically generated - may contain errors
Lab products found in correlation
Lipopolysaccharides (lps), by Merck Group (2 mentions)
Celltrace violet, by Thermo Fisher Scientific (2 mentions)
Anti cd3 mab, by BioLegend (2 mentions)
Easysep treg isolation kit, by STEMCELL (2 mentions)
Recombinant il 10, by Thermo Fisher Scientific (2 mentions)
Anti il 10, by BioLegend (2 mentions)
Tofacitinib, by Apexbio (1 mentions)
Tocilizumab, by Selleck Chemicals (1 mentions)
Anti ifnar2, by PBL Assay Science (1 mentions)
Rosiglitazone, by Cayman Chemical (1 mentions)
Troglitazone, by Cayman Chemical (1 mentions)
Pioglitazone, by Cayman Chemical (1 mentions)
Gw9662, by Cayman Chemical (1 mentions)
Mouse igg1 isotype control, by BioLegend (1 mentions)
Rat igg2a isotype control, by BioLegend (1 mentions)
Il 10, by Thermo Fisher Scientific (1 mentions)
Pam3csk4, by Thermo Fisher Scientific (1 mentions)
Bead based multiplex immunoassay, by Bio-Rad (1 mentions)
7 protocols using anti il 10r
1
Regulatory T Cell Suppression of Lupus Nephritis
2
Measuring Treg-mediated Suppression with miR-106a
3
Whole Blood Cytokine Modulation Assay
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Whole blood was incubated for 4 h with tofacitinib (500 nM, ApexBio) or tocilizumab (50 μg ml−1, Selleckchem) or vehicle controls (DMSO and human IgG control, 50 μg ml−1, BioLegend, respectively). For IFN-I blocking, whole blood was incubated with a combination of anti-IFNAR2 (2.5 μg ml−1 PBL Assay Science), anti-IFNα (0.2 μg ml−1, PBL 31110–1) and IFNβ (0.2 μg ml−1, PBL 31401-1) antibodies or vehicle control for 4 h. For IL-10 blocking, whole blood was incubated with a combination of anti-IL-10 (5 μg ml−1, BioLegend) and anti-IL-10R (5 μg ml−1, BioLegend) for 4 h. After blocking, whole blood was fixed using Proteomic Stabilizer PROT1 (1.4× blood volume, SmartTube) for 10 min at room temperature and frozen at −80 °C.
4
Pioglitazone Modulates Sepsis Response
5
IL-10+ Extracellular Vesicles Protect Cells from Hypoxia-Reoxygenation Injury
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TECs were plated in 35-mm dishes at a density of 1 × 106 cells per dish and incubated until they reached approximately 80 to 90% confluence for experiment. The cells in H/R group were cultured for 12 hours under hypoxic conditions (94% N2 and 5% CO2) in medium without nutrients (glucose-free, FBS-free). In addition, cells were then transferred back to regular culture medium with oxygen for 2 hours of reoxygenation. Control cells were incubated in complete culture medium in a regular incubator (5% CO2 and 95% air). For the IL-10+ EV treatment, cells were incubated with IL-10+ EVs (5 and 15 μg) or together with anti-IL10R (10 μg/ml; clone: 1B1.3a, BioLegend) for 12 hours during hypoxia.
6
Modulation of Dendritic Cell Cytokine Production
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FACS-sorted mDCs (2×105/well) were incubated with 20 ng/ml TSLP (R&D Systems, 1398-TS-010/CF) (12 (link)), 100 μg/ml endotoxin-free curdlan (FUJIFILM Wako Pure Chemical Corporation) (24 (link)), 20 μg/ml (133 nM) anti-human Dectin-1 monoclonal antibody (anti-hDectin-1 mAb, clone 15E2) (18 (link)), 133 nM Pam3CSK4 (Invitrogen) or 133 nM anti-hDectin-1-Pam3CSK4 conjugate (described below) for indicated time. Cytokine concentration in the supernatants was measured by a bead-based multiplex immunoassay (Bio-Rad) according to manufacturer’s instruction. In cytokine blocking experiments, 10 μg/ml anti-IL-10 (BioLegend, 506813), anti-IL-10R (BioLegend, 308817), isotype control rat IgG2a (BioLegend, 400543), anti-TNFα (R&D Systems, MAB210-100) or isotype control mouse IgG1 (BioLegend, 400166) was added into culture 1h or 3h before stimulation. In some experiments, 1 μg/ml recombinant human TNFα (R&D Systems, 210-TA/CF) or IL-10 (PeproTech, 200-10) was added into culture. For inhibition assay, 10 μM STAT3 inhibitor Stattic (25 (link)) (Sigma-Aldrich, S7947-25MG), STA-21 (26 (link)) (Cayman Chemical, 14996) or Syk inhibitor R406 (24 (link), 27 (link)) (Invivogen, inh-r406) was used to inhibit kinase activity as indicated. DMSO was used as vehicle control at 0.1%.
7
Measuring Treg-mediated Suppression with miR-106a
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Treg-mediated suppression was measured as previously described 37 (link). Briefly, CD4+CD25+ Tregs were isolated by negative selection of CD4+ T cells, followed by positive selection of CD25+ cells using the EasySep Treg isolation kit (STEMCELL). Treg frequency was previously confirmed by flow cytometry with approximately 85% of enriched CD4+CD25+ expressing FoxP3 38 . CD4+CD25Neg effector T cells were labeled with CellTrace Violet (Invitrogen) according to manufacturer’s instructions prior to stimulation with anti-CD3 mAb (1μg/ml; 145–2C11, BioLegend) in the presence of irradiated syngeneic APC to allow for fluorescent monitoring of cell proliferation. Proliferating cells were co-cultured with varying ratios of converted CD4+CD25+ Treg from WT or TNFΔARE/+ which had been transduced with either miRZIP000 or miRZIP106a to determine the impact of selective inhibition of miR-106a on Treg suppressive function. Anti-IL-10R (10μg/ml; 1B1.3a, BioLegend) or recombinant IL-10 (10ng/ml; Peprotech, Rocky Hill, NJ) were added to culture conditions as indicated.
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