After culturing overnight, explants were incubated with 1, 10 or 100 ng/ml rhIL-37 (R&D) in medium without serum for up to 48 h or 6 days, with new addition of rhIL-37 every other day. Additionally, inhibitors against MMP-3 (1 mM, Batimastat CAS 130370-60-4, MedChem Express), MMP-13 (1 mM, CAS 544678-85-5, Calbiochem) or ADAMTS-5 (50 mM, CAS 929634-33-3, EMD Millipore) were added following the same protocol. The functionality of these inhibitors on GAGs release was first confirmed, see Supplementary Fig. 2.
Rhil 37
Manufactured by R&D Systems
Sourced in United States
RhIL-37 is a recombinant human interleukin-37 protein. Interleukin-37 is a member of the interleukin-1 cytokine family and is believed to function as a broad-spectrum inhibitor of innate inflammatory and immune responses.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin, by Thermo Fisher Scientific (3 mentions)
Streptomycin, by Thermo Fisher Scientific (3 mentions)
Nex041h001mc, by PerkinElmer (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Rhil 6, by Thermo Fisher Scientific (1 mentions)
Rhil 10, by Thermo Fisher Scientific (1 mentions)
Rh il 22, by Thermo Fisher Scientific (1 mentions)
Anti il 6, by Thermo Fisher Scientific (1 mentions)
Anti tnf α, by Thermo Fisher Scientific (1 mentions)
Anti il 1β, by Thermo Fisher Scientific (1 mentions)
Sh 4 54, by Selleck Chemicals (1 mentions)
Rhil 1β, by Thermo Fisher Scientific (1 mentions)
Rhtnf α, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Ovalbumin (ova), by R&D Systems (1 mentions)
Ovalbumin (ova), by Merck Group (1 mentions)
8 protocols using rhil 37
1
Regulation of Extracellular Matrix Metabolism
2
Cytokine-Mediated Immune Modulation in PBMC
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PBMC from healthy donors were cultured in complete medium (RPMI 1640 supplemented with 10% heat-inactivated FBS, 100 µ/mL penicillin and 100 µg/mL streptomycin [Life Technologies]) at a concentration of 1 × 106 cells/mL for 3, 5, and 7 days with the plasma of HBV-ACLF and NC, or various concentrations (10–50 ng/mL) of recombinant human (rh) interleukin rhIL-1β (PeproTech), rhIL-6 (PeproTech), rhIL-10 (PeproTech), rhIL-22 (PeproTech), rhIL-37 (R&D), and tumor necrosis factor (rhTNF-α, PeproTech), or 10 μg/mL neutralizing antibody of anti-IL-6 (PeproTech), anti-TNF-α (PeproTech), and anti- IL-1β (PeproTech). The medium and cytokines were refreshed every other day. The expression of BTLA in CD4+ T cells was analyzed using flow cytometry.
PBMC from healthy donors were cultured at a concentration of 1 × 106 cells/mL in complete medium and were treated for 3 days in the following groupings: (a) control; (b) stimulant alone: rhIL-6 alone (50 ng/mL), or rhTNF-α (50 ng/mL); (c) inhibitor alone: anti-stat3 (SH-4-54, 50 μmol/L, Selleck) or the anti-NF- kappa (QNZ, 20 μmol/L, Selleck) or Polymyxin B (PXB, 10 μg/mL, Sigma-Aldrich); and (d) stimulant plus inhibitors. All experiments were performed at least in duplicate.
PBMC from healthy donors were cultured at a concentration of 1 × 106 cells/mL in complete medium and were treated for 3 days in the following groupings: (a) control; (b) stimulant alone: rhIL-6 alone (50 ng/mL), or rhTNF-α (50 ng/mL); (c) inhibitor alone: anti-stat3 (SH-4-54, 50 μmol/L, Selleck) or the anti-NF- kappa (QNZ, 20 μmol/L, Selleck) or Polymyxin B (PXB, 10 μg/mL, Sigma-Aldrich); and (d) stimulant plus inhibitors. All experiments were performed at least in duplicate.
3
Modulation of gMDSC by Inflammatory Cytokines
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PBMCs from healthy donors were cultured in a complete medium at 1 × 106 cells/mL for 1, 3, 5, and 7 days with either HBV-ACLF and HC plasma, or various concentrations (5–50 ng/mL) of recombinant human (rh) tumor necrosis factor (rhTNF-α), interleukin-6 (rhIL-6), rhIL-1β, rhIL-22, rhIL-37, rhIL-10, and rhFGF2. All antibodies were purchased from PeproTech, and rhIL-37 was purchased from R&D Systems. The medium and cytokines were refreshed every other day. Flow cytometry was used to determine the number of gMDSC.
Next, PBMCs from healthy donors were cultured in complete medium and split into the following groups: (a) control (no treatment); (b) stimulant alone (50 ng/mL): rhIL-6 or rhTNF-α alone; (c) inhibitor alone: anti-TNF-α (10 μg/mL, eBscience), stat3 inhibitor (SH-4-54, 50 μmol/L, Selleck), or NF-kb inhibitor (QNZ, 20 μmol/L, Selleck); and (d) stimulant plus inhibitors. Each treatment lasted for 3 days. All experiments were performed six times.
Next, PBMCs from healthy donors were cultured in complete medium and split into the following groups: (a) control (no treatment); (b) stimulant alone (50 ng/mL): rhIL-6 or rhTNF-α alone; (c) inhibitor alone: anti-TNF-α (10 μg/mL, eBscience), stat3 inhibitor (SH-4-54, 50 μmol/L, Selleck), or NF-kb inhibitor (QNZ, 20 μmol/L, Selleck); and (d) stimulant plus inhibitors. Each treatment lasted for 3 days. All experiments were performed six times.
4
IL-37 Modulation of PBMC Cytokine Response
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Whole-blood PBMCs were cultured in RPMI 1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) with 100 μg/ml streptomycin (Thermo Fisher Scientific), 100 IU/ml penicillin, and 10% FBS (Thermo Fisher Scientific) as culture medium in a humidified atmosphere of 5% CO2 at 37 °C. PBMCs were stimulated with or without recombinant human IL-37 (rhIL-37, catalogue number 1975-IL-025; R&D Systems, Minneapolis, MN, USA) at various concentrations for 24 h, then incubated further with 1 μg/ml LPS (Sigma-Aldrich, St. Louis, MO, USA) for 4 h. Total RNA was extracted [42 (link)], and cytokine transcription was analyzed by qRT-PCR. To determine cytokine protein expression in PBMCs, cells were stimulated with or without rhIL-37 at 100 ng/ml for 24 h, then incubated further with LPS (1 μg/ml) for 8 h, and culture supernatants were harvested and frozen at − 80 °C for later cytokine analysis by enzyme-linked immunosorbent assay (ELISA).
5
Intranasal OVA-Induced Asthma Model
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Experiments were approved by the China-Japan Friendship Hospital Animal Experimental Ethics Committee in Beijing, China. 6-8 weeks female BALB/c were procured from Vital River Laboratory Animal Technology Co. Ltd (China) and were housed in a pathogen-free environment in clinic research institute of China-Japan Friendship Hospital, Beijing, China. Mice were kept in a 12-hour light-dark cycle with ad libitum access to food and water.
Thirty mice were randomly categorized into three groups i.e., the Control, OVA/PBS, and OVA/IL-37 groups. In OVA/PBS group, the mice were sensitized by intraperitoneal injection of OVAlbumin (OVA, Sigma-Aldrich, Beijing, 100ug emulsified in Al (OH)3 /dose) on day 0, day7, day14, then further challenged every other day per-nasally from day 22 to day 30 with 100ug of OVA in 50uL PBS/dose. In the Control group, an equal amount of Al (OH)3 (intraperitoneally) was given to mice and then nasally challenged with PBS at the same time points as the actively challenged mice (Figure 1A ). The mice in OVA/IL-37 group were treated with rhIL-37 (R&D System, USA, 200ng/dose) or with PBS as control 24h before OVA administration.
Thirty mice were randomly categorized into three groups i.e., the Control, OVA/PBS, and OVA/IL-37 groups. In OVA/PBS group, the mice were sensitized by intraperitoneal injection of OVAlbumin (OVA, Sigma-Aldrich, Beijing, 100ug emulsified in Al (OH)3 /dose) on day 0, day7, day14, then further challenged every other day per-nasally from day 22 to day 30 with 100ug of OVA in 50uL PBS/dose. In the Control group, an equal amount of Al (OH)3 (intraperitoneally) was given to mice and then nasally challenged with PBS at the same time points as the actively challenged mice (
6
Optimizing rhIL-37 and PFK15 in A549 Cells
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A549 cells were treated with different concentrations (0 ng/ml, 10 ng/ml, 50 ng/ml, 100 ng/ml, 200 ng/ml) of rhIL-37 (R&D Systems, Minneapolis, MN, USA)for different times (24 h, 48 h, 72 h) to explore the optimal concentration and time; 1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one(PFK15) (Sigma-Aldrich, St. Louis, MO, USA) treat A549 cells with different concentrations (0 µM, 2 µM, 4 µM, 6 µM, 8 µM, 10 µM) for 24 h to find the optimal concentration.
7
Cartilage Proteoglycan Production Assay
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Explants were placed in 1 ml of incubation medium consisting of DMEM/HAMS-F12 (1:1) without serum and incubated with rhIL-37 (R&D) for 48 h. Subsequently, cartilage explants were labeled with 3.7 Â 10 5 Bq 35 S-sulphate ( 35 S, NEX041H001MC, PerkinElmer). After labeling for 4 h at 37 C, the explants were rinsed thrice in saline and dissolved in Luma Solve (Hicol, Oud-Beijerland, The Netherlands) overnight at 60 C. The 35 S-sulphate content of the explants, which is a reliable measure of cartilage proteoglycan production 28 , was measured in Lipoluma (Hicol, Oud-Beijerland, The Netherlands) by liquid scintillation counting.
8
Measuring Cartilage Proteoglycan Degradation
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To study the in vitro degradation of human OA cartilage, cartilage explants were pre-labeled with 3.7 Â 10 5 Bq 35 S-sulphate radionuclide ( 35 S, NEX041H001MC, PerkinElmer) for 4 h at 37 C. Subsequently, the explants were rinsed thrice with saline, cultured overnight and incubated with rhIL-37 (R&D) for 48 h. Next, explants were dissolved in Luma Solve (Hicol, Oud-Beijerland, The Netherlands) overnight at 60 C. The amount of 35 S-sulphate labeled proteoglycans was determined by liquid scintillation counting.
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