IL-1β (211-11B; PeproTech, Rocky Hill, NJ, USA) was dissolved in DKSFM to prepare stocks with a concentration of 10 mg/mL. Rosiglitazone (HY-17386; MedChemExpress, Monmouth, Junction, NJ, USA) was dissolved in DKSFM to prepare stocks with a concentration of 5 mM. HMGDCs were exposed to medium containing 50 ng/mL IL-1β, 50 ng/mL IL-1β + 50 µM Rosiglitazone, or DKSFM alone for a duration of 48 hours, when the cells reached approximately 60% confluency. These concentrations of pharmacologic reagents were selected based on previous studies conducted on mouse MG explants. The earlier investigations revealed that the treatment of mice MGs with 50 ng/mL IL-1β did not yield any noteworthy impact on cell activity, while a noticeable keratinizing effect was observed.8 ,9 (link)
Il 1β
Manufactured by MedChemExpress
Sourced in United States, China
IL-1β is a recombinant human interleukin-1 beta protein. Interleukin-1 beta is a pro-inflammatory cytokine that plays a key role in the regulation of immune and inflammatory responses.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (4 mentions)
Il 1β, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Merck Group (2 mentions)
Penicillin, by Merck Group (2 mentions)
Tgf β1, by MedChemExpress (2 mentions)
Mg132, by Selleck Chemicals (2 mentions)
Chloroquine cq, by MedChemExpress (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Rosiglitazone, by MedChemExpress (1 mentions)
Il 1β, by Merck Group (1 mentions)
Nigericin, by MedChemExpress (1 mentions)
Propionate, by Merck Group (1 mentions)
Butyrate, by Merck Group (1 mentions)
Panobinostat, by Merck Group (1 mentions)
M csf, by R&D Systems (1 mentions)
Rankl, by R&D Systems (1 mentions)
Bafilomycin a1 baf a1, by MedChemExpress (1 mentions)
Mrc 5, by American Type Culture Collection (1 mentions)
15 protocols using il 1β
1
Effect of IL-1β and Rosiglitazone on HMGDCs
2
Senkyunolide A Mitigates IL-1β-Induced Chondrocyte Inflammation
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Chondrocytes were stimulated with IL-1β (Xu and Xu 2017 (link)) (10 ng/mL, Sigma-Aldrich, St. Louis, MO) for 24 h to simulate an inflammatory response. For the SenA treatment groups, chondrocytes were pre-treated with different concentrations of SenA (0, 20, 40, 80 and 160 μg/mL, MedChemExpress, Monmouth Junction, NJ) for 2 h in serum free medium. To investigate the effects of SenA on IL-1β-induced chondrocytes, the chondrocytes were divided into five groups: control group (untreated cells), IL-1β, IL-1β + SenA 20, IL-1β + SenA 40 μg/mL group and IL-1β + SenA 80 μg/mL group. To confirm the effects of SenA on IL-1β-stimulated chondrocytes through the NLRP3 signalling pathway, the NLRP3 activator, nigericin (Nig, 15 μmol/L, MedChemExpress, Monmouth Junction, NJ) was used to treat chondrocytes. Chondrocytes were divided into four groups: control group, IL-1β group, IL-1β + SenA (80 μg/mL) group and IL-1β + SenA + Nig group. SenA was dissolved in 0.1% dimethyl sulphoxide (DMSO). In all cell cultures, the concentration of DMSO was less than 0.05%, which had no perceptible impact on cell growth or death.
3
Osteoclast Differentiation from Bone Marrow Cells
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Total bone marrow cells from WT and GPR109A−/− mice were isolated by flushing the femora and tibiae. The cells were plated overnight at 37 °C with 5.5% CO2 in alpha minimum essential medium (αMEM), supplemented with 10% FBS and 1% penicillin/streptomycin (Gibco, Carlsbad, CA, USA), and 40 ng/ml macrophage colony stimulating factor (M-CSF; R&D Systems). Next day, nonadherent cells were collected and cultured in osteoclast medium with 40 ng/ml M-CSF and 50 ng/ml receptor activator of nuclear factor kappa-B (NF-κB) ligand (RANKL; R&D Systems) in 24-well plates (for tartrate-resistant acid phosphatase (TRAP) staining, immunofluorescence), 12-well plates (for RNA isolation), or 6-well plates (for Western blotting) at a density of 1 × 106 cells/ml at 37 °C and 5.5% CO2. The medium was changed every 3 d. In SCFA experiments, preosteoclasts in C3 and C4 groups were further stimulated on day 1 of culture with the following stimulants: 5 mmol/L propionate, 1 mmol/L butyrate (Sigma-Aldrich), 40 ng/ml IL-1β (MedChemExpress), 50 nmol/L TSA (MedChemExpress), 25 nmol/L Panobinostat (Sigma-Aldrich), 100 μmol/L 4-CMTB (Sigma-Aldrich), 1 mmol/L niacin (MedChemExpress), or 25 μmol/L AR42062 (Sigma-Aldrich). Then, fully differentiated osteoclasts (days 5–7) were collected for PCR, Western blotting, TRAP staining, and immunofluorescence staining.
4
Induction of RA-ILD Cell Model
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The human fetal lung fibroblast cell line (MRC-5) was purchased from American Type Culture Collection (ATCC, Rockville, MD, USA) and cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Life Technologies/Gibco, Grand Island, NY, USA) supplemented with 10 % (v/v) fetal bovine serum (Gibco) and 1% antibiotic-antimycotic solution (containing 100 U/mL penicillin, 100 µg/mL streptomycin, and 0.25 µg amphotericin B; Sigma-Aldrich, St. Louis, MO, USA). Cells were maintained in a humidified air atmosphere with 5% CO2 at 37 °C.
To induce the cell model of RA-ILD along with the animal model, recombinant TGF-β1 and IL-1β were added to the cell medium for 24 h, purchased from MedChemExpress (Monmouth Junction, NJ, USA). Chloroquine (CQ) and Bafilomycin A1 (BafA1) were purchased from MedChemExpress (Monmouth Junction, NJ, USA). MG132 was purchased from Selleckchem (Houston, TX, USA).
To induce the cell model of RA-ILD along with the animal model, recombinant TGF-β1 and IL-1β were added to the cell medium for 24 h, purchased from MedChemExpress (Monmouth Junction, NJ, USA). Chloroquine (CQ) and Bafilomycin A1 (BafA1) were purchased from MedChemExpress (Monmouth Junction, NJ, USA). MG132 was purchased from Selleckchem (Houston, TX, USA).
5
Paroxetine Modulates RANKL-Induced Osteoclastogenesis
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Paroxetine was purchased from Apexbio (Suzhou, China). Alpha-MEM and DMEM were procured from Biosharp (Anhui, China). The IL-1β and Cell Counting Kit-8 (CCK-8) were purchased from Med Chem Express (New Jersey, USA). RANKL and M-CSF were procured from LifeTein (Beijing, China). Primary antibodies against Nlrp3 (ab263899, 1:1000), IL-1β (ab254360, 1:1000), Caspase1 (ab179515, 1:1000) and ADAMTS5 (ab185722, 1:1000) were purchased from Abcam (Cambridge, UK). The aggrecan antibody (PA1-1746, 1:1000) was procured from Thermo Fisher Scientific (Waltham, MA, USA), and MMP3 (ET1705-98, 1:1000) and SOX9 (ET1611-56, 1:1000) antibodies were purchased from Huaan Biotechnology (Hangzhou, China). Primary antibodies against P65 (8242, 1:1000), p-P65 (3033, 1:1000), IκBα (4812, 1:1000), p-IκBα (2859, 1:1000), and β-actin (4970, 1:10,000) were procured from Cell Signaling Technology (Danvers, MA, USA). The NFATc1 antibody (YT5381, 1:1000) was procured from ImmunoWay (Shanghai, China).
6
Synovial Fibroblasts in Osteoarthritis
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The study was designed according to the Declaration of Helsinki, and was approved by Ethic Committee of the Second Affiliated Hospital of Harbin Medical University (KY 2021-256). Informed consent was obtained from each donor. Synovium of 3 OA patients (age 54–70 years, Kellgren-Lawrence grade 4) that underwent total joint arthroplasty (TKA) and 3 patients (age 56–68 years) that underwent meniscectomy without OA were obtained at the time after surgery. All patients were confirmed without Rheumatoid Arthritis, acute trauma, tumor or infection of knee joint. Briefly, synovium was cut into pieces at the final size about 0.5 mm*0.5 mm, and put into 0.1% type I collagenase (Biosharp, China, BS163). α-MEM medium (Cytiva, United States, SH30265.01) was added with 10% foetal bovine serum (ExCell Bio, China, FSD500) after 2 h. Primary cells could be seen climbing out after about 3–5 days. FLS of passage 6-8 (P6-8) were used in this study. 10 ng/ml of IL-1β (PEPROTECH, United States, 200-01B) were used to stimulate FLS of OA groups for 48 h in order to imitate the environment of OA, while complete medium was added into the FLS of control group. 10 μg/ml of Etanercept and Iguratimod (MedChem Express, China, HY-108847, HY-17009) were added along with IL-1β to FLS for the following test.
7
Murine Macrophage Culture and Stimulation
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The mouse peritoneal macrophages (MPMs) extracted from C57BL/6 female mice and MH-S cells and were cultured routinely in Roswell Park Memorial Institute (RPMI) medium (HyClone) supplemented with 10% fetal bovine serum (FBS), l -glutamine, and penicillin. Immortalized bone marrow-derived macrophage (iBMDM) cells and Raw264.7 cells were cultured routinely in Dulbecco’s modified Eagle’s medium (DMEM) (HyClone) supplemented with 10% FBS, l -glutamine, and penicillin. All cells were cultured in antibiotic-free medium before PA stimulation or inhibitor pretreatment. The cells were pretreated with VX-765 (HY-13205, Med Chem Express), Nig (HY-13205, Med Chem Express), IL-1β (HY-P7073, Med Chem Express), IL-1β receptor inhibitor (TLR-1, HY-W011400, Med Chem Express), AKT inhibitor VIII (HY-10355, Med Chem Express), or IFN-β (CAT 300-02BC, PeproTech) for the indicated times before stimulation with PA, as indicated; multiplicity of infection (MOI) values are given for the different times.
8
Chondroprotective Effects of PDGF, IL-1β, and Imatinib
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RHu PDGF-BB, IL-1β and imatinib mesylate were purchased from MedChemExpress, United States (cat. nos. HY-P7055; HY-P7097; HY-50946); PDGF-BB polyclonal antibody, collagen II monoclonal antibody and aggrecan neo polyclonal antibody were purchased from Thermo Fisher Scientific, United Kingdom (cat. nos. PA5-88272; MA5-12789; PA1-1746); MMP3 antibody, MMP9 antibody, Adamts4 antibody, Adamts5 antibody and Caspase1 antibody were purchased from Abcam, United Kingdom (cat. nos. ab52915; ab76003; ab185722; ab41037; ab179515); PDGFR antibody, P-PDGFR antibody, P-P38 antibody and P-JNK antibody were purchased from Hangzhou Huaan Biotechnology Co., Ltd., China (cat. nos. SN0646; R1510-44; ER2001-52; ET1601-28); P-PI3K antibody, PI3K antibody, P-AKT antibody, AKT antibody, P38 antibody, JNK antibody, P-ERK antibody, ERK antibody, β-actin antibody and NLRP3 antibody were purchased from Cell Signaling Technology, United States (cat. nos.17366; 4292; 9271; 4691; 9212S; 9252S; 4370; 4695; 4970; ab263899); IL-1β antibody was purchased from ABclonal Technology Co., Ltd., China (cat. no. A1112).
9
Establishing DM-OA Synovial Fibroblast Model
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To establish DM‐OA cell model in vitro, rat synovial fibroblasts were purchased from iCell Bioscience Inc. (Shanghai, China) and maintained in DMEM medium (Gibco Life Technologies, Carlsbad, CA, USA) supplemented with 1% fibroblast growth supplement (Sciencell, Carlsbad, CA, USA) and 10% fetal bovine serum (Sigma‐Aldrich, St Louis, MO, USA). Cells were infected with lentivirus particles to overexpress PSTPIP2. Two days after lentivirus infection, cells were added with normal glucose (5.5 mmol/L, Aladdin regents, Shanghai, China), high glucose (25 mmol/L), IL‐1β (5 ng/mL, MedChemExpress, Monmouth Junction, NJ, USA) or ERK inhibitor U0126 (10 μM, MedChemExpress) according to the group information. Two days later, cells were collected for further testing.
10
Chondrocyte Apoptosis and OA Modulation
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Human primary articular chondrocytes (Saibaikang Biotechnology Co.) were cultured in DMEM (Gibco) containing 10% fetal bovine serum (Gibco) and 1% streptomycin/penicillin antibiotics, and maintained in a humidified incubator under 5% CO2 at 37°C. Interleukin-1β (IL-1β) is an important pro-inflammatory cytokine that can induce chondrocyte apoptosis, leading to cartilage matrix degradation and joint inflammation, and thus, promoting the progression of OA [33 , 34 ]. Therefore, the chondrocytes were exposed to 10 ng/mL IL-1β (Med Chem Express, Monmouth Junction, NJ, USA) for 24 h to construct the OA cell model [35 ].
The RAD54L-overexpression plasmid (pcDNA3.1-RAD54L) was acquired from Shenggong Bioengineering Company (Shanghai, China). Cells induced with IL-1β were transfected with pcDNA3.1-RAD54L or the empty plasmid, pcDNA3.1-NC, using Lipofectamine 3000 (Life Technologies Corporation, Carlsbad, CA, USA) for 48 h to overexpress RAD54L (IL-1β+oe-RAD54L group) or as a control (IL-1β+oe-NC group), respectively. Therefore, the cells were divided into 4 groups: Control (chondrocytes without any treatment), IL-1β, IL-1β+oe-NC, and IL-1β+oe-RAD54L groups.
The RAD54L-overexpression plasmid (pcDNA3.1-RAD54L) was acquired from Shenggong Bioengineering Company (Shanghai, China). Cells induced with IL-1β were transfected with pcDNA3.1-RAD54L or the empty plasmid, pcDNA3.1-NC, using Lipofectamine 3000 (Life Technologies Corporation, Carlsbad, CA, USA) for 48 h to overexpress RAD54L (IL-1β+oe-RAD54L group) or as a control (IL-1β+oe-NC group), respectively. Therefore, the cells were divided into 4 groups: Control (chondrocytes without any treatment), IL-1β, IL-1β+oe-NC, and IL-1β+oe-RAD54L groups.
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