Recombinant IL-1β, forskolin and the inhibitors LY294002, U0126, SB203580 and SP600125 were obtained from Sigma-Aldrich Corp. Antibodies specific for β-actin, AKT, p-AKT (Ser 473), ERK1/2, p-ERK1/2 (Thr 202/Tyr 204), p38, p-p38 (Thr 180/Tyr 182), c-Jun, p-c-Jun (Ser 63), NF-κB, p-NF-κB (Ser 536), p-NF-κB (Ser 276), IL-1β and siRNAs targeting AKT, ERK1/2, p38 were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Quinazoline (QNZ) was obtained from Enzo Life Sciences (Farmingdale, NY, USA). The IL-1β and cAMP enzyme immunoassay kits were from Cayman Chemical (Ann Arbor, MI). Recombinant human MMP-7 was obtained from R&D systems (Minneapolis, MN, USA). MMP-7 cDNA plasmids were purchased from Origene Technologies (Rockville, MD, USA). All reagents for qRT-PCR and SDS-PAGE experiments were purchased from Bio-Rad Laboratories. All other reagents were from Invitrogen (Carlsbad, CA) unless otherwise specified.
Recombinant il 1β
Manufactured by Merck Group
Sourced in United States
Recombinant IL-1β is a laboratory product that functions as a cytokine. It is a recombinant form of the interleukin-1 beta protein, which is involved in various immunological and inflammatory processes.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
High performance liquid chromatography (hplc), by Merck Group (2 mentions)
Dmem f12 medium, by Merck Group (2 mentions)
Penicillin, by Merck Group (2 mentions)
Streptomycin, by Merck Group (2 mentions)
6 well plate, by Corning (2 mentions)
Sb203580, by Merck Group (1 mentions)
Forskolin, by Merck Group (1 mentions)
Quinazoline qnz, by Enzo Life Sciences (1 mentions)
P nf κb, by Cell Signaling Technology (1 mentions)
Sp600125, by Merck Group (1 mentions)
Il 1β, by Cell Signaling Technology (1 mentions)
P c jun, by Cell Signaling Technology (1 mentions)
U0126, by Merck Group (1 mentions)
Ly294002, by Merck Group (1 mentions)
Pbind gr, by Promega (1 mentions)
Bright glo, by Promega (1 mentions)
Ru486, by Merck Group (1 mentions)
Nuclear extract kit, by Active Motif (1 mentions)
Hc a cells, by Merck Group (1 mentions)
8 protocols using recombinant il 1β
1
Investigating Inflammatory Signaling Pathways
2
Purification and Characterization of TCDCA
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TCDCA was dissociated and depurated from chicken bile as described in our previous study [7 (link),8 (link)], with the purity being> 99.5%. RU486 and recombinant IL-1β were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Freund′s complete adjuvant (FCA) was purchased from the Shanghai Institute of Biological Products (Shanghai, China). The Nuclear Extract Kit and TransAM® c-Jun activation kit were purchased from Active Motif (Carlsbad, CA, USA). Phenol red-free Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Life Technologies (Waltham, MA, USA). pGL4.35 [luc2P/9 × GAL4UAS/Hygro], pBIND-GR and Bright-Glo™ the luciferase assay system were purchased from Promega (Madison, WI, USA).
3
Chondrogenic cell culture and IL-1β treatment
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The human chondrogenic HC-A cells (ATCC, USA) were cultured in DMEM/F12 medium (Sigma-Aldrich, St. Louis, MO) supplemented with 5% FBS (Gibco, Grand Island, NY), 100 U/mL penicillin (Sigma-Aldrich), and 100 μg/mL streptomycin (Sigma-Aldrich) in a humidified incubator with 95% air and 5% CO2 at 37 °C. The cells were routinely cultured in in 6-well plates (Corning, New York, NY) with a density of 6 × 104/well. The recombinant IL-1β with purity greater than 98% (HPLC) was purchased from Sigma-Aldrich. Cells were treated by 5 μg/mL IL-1β for 24 h.
4
Mimicking Tendinopathy with IL-1β and iMSC-sEVs
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IL-1β is significantly upregulated in tendinopathy tissue and is used to mimic conditions in vitro.42 (link),43 (link) Briefly, before the stimulation of IL-1β, tenocytes were cultured in a medium containing 1% serum for 24h. The medium was then replaced with the medium containing 100 ng/mL recombinant IL-1β (Sigma, St. Louis, MO, USA), 100 ng/mL IL-1β and 1×109 p/mL iMSC-sEVs. The tenocytes were divided into the following three groups (n = 3 per group) according to different treatments: control group (PBS), IL-1β+vehicle group (100 ng/mL IL-1β), and IL-1β+sEVs group (100 ng/mL IL-1β + 1×109 p/mL iMSC-sEVs).
5
Signaling Pathway Analysis Techniques
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The following antibodies were used: NF-κB p65 (western blot 1:1,000, rabbit, Cell Signaling, #8284), GAPDH (Western blot 1:3,000, mouse, Santa Cruz, #sc-47724), phospho (Ser 473) Akt (Western blot 1:1,000, rabbit, Cell Signaling, #9271), Akt (Western blot 1:1,000, rabbit, Cell Signaling, #9272), phospho (Ser21/9) GSK-3α/β (Western blot 1:1,000, rabbit, Cell Signaling, #9331), GSK-3α/β (Western blot 1:1,000, mouse, Santa Cruz, #sc-7291), phospho (Ser675) β-catenin (Western blot 1:1,000, rabbit, Cell Signaling, #4176), β-catenin (Western blot 1:1,000, mouse, BD Biosciences, #610154), Histone H3 (Western blot 1:1,000, rabbit, #4499), non-phospho (active) β-catenin (Western blot 1:500, rabbit, Cell Signaling, #8814), IκB-α (Western blot 1:1,000, rabbit, Santa Cruz, #sc-203), acetyl NF-κB p65 (Lys310) (Western blot 1:500, rabbit, Cell Signaling, #3045), and normal mouse IgG (Santa Cruz, #sc-2025).
Other reagents include the following: DMSO (Sigma Aldrich, #472301), ICG-001 (Tocris, #4505), XAV-939 (Tocris, #3748), IQ-1 (Tocris, #4713), FBS (Thermo Scientific, #SV30180.03), and recombinant IL-1β (Sigma Aldrich, #SRP3083). All other chemicals were of analytical grade.
Other reagents include the following: DMSO (Sigma Aldrich, #472301), ICG-001 (Tocris, #4505), XAV-939 (Tocris, #3748), IQ-1 (Tocris, #4713), FBS (Thermo Scientific, #SV30180.03), and recombinant IL-1β (Sigma Aldrich, #SRP3083). All other chemicals were of analytical grade.
6
Chondrogenic ATDC5 Cells Treated with IL-1β
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The human chondrogenic ATDC5 cells (ECACC, Porton Down, Wiltshire, UK) were cultured in DMEM/ F12 medium (Sigma-Aldrich, St. Louis, MO) supplemented with 5% FBS (Gibco, Grand Island, NY), 100 U/ mL penicillin (Sigma-Aldrich), and 100 μg/mL streptomycin (Sigma-Aldrich) in a humidified incubator with 95% air and 5% CO 2 at 37°C. The cells were routinely cultured in in 6-well plates (Corning, New York, NY) with a density of 6 × 10 4 /well.
The recombinant IL-1β with purity greater than 98% (HPLC) was purchased from Sigma-Aldrich. Cells were treated by 1 ng/mL IL-1β for 12 h, as previously described [11] .
The recombinant IL-1β with purity greater than 98% (HPLC) was purchased from Sigma-Aldrich. Cells were treated by 1 ng/mL IL-1β for 12 h, as previously described [11] .
7
Primary Mouse Hepatocyte Isolation and Culture
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Anesthetized 8-week old WT mice were perfused via the inferior vena cava as previously described [26] (link). Briefly, mice were perfused with a 0.5 mM EGTA solution followed by enzymatic collagenase digestion (Sigma) in 1 mM CaCl2. Hepatocytes were washed with 1 mM CaCl2 (3x) and separated by centrifugation. Primary hepatocytes were seeded in 6-well plates. Before starting stimulation experiments, hepatocytes were rested for 3 hours in M199 media containing 1% Penicillin/Streptomycin, 10% FA-free BSA, 2% FBS, Dexamethasone (100 uM), and Insulin (100 nM) at 37°C and 5% CO2. Subsequently, culture media was replaced and cells were maintained in M199 media containing Penicillin/Streptomycin, Dexamethasone and Insulin. Insulin was present in all experimental media to maintain the health of the primary cultures. Cells were left untreated or treated with 10 ng/ml recombinant IL-1β (Millipore) for 24 hours.
8
Endothelial Cell Culture and Inflammation
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HUVEC (PromoCell, Heidelberg, Germany) and microvascular adult human dermal blood endothelial cells (HDBEC) from adult skin (PromoCell) were cultured in endothelial cell growth medium MV (PromoCell) supplemented with 5 % (v/v) foetal calf serum (FCS) and endothelial cell medium MV growth supplements (endothelial cell growth supplement (0.004 mg/ml), recombinant human epidermal growth factor (10 ng/ml), heparin (90 µg/ml) and hydrocortisone (1 µg/ml)). Cells were cultured at 37˚C under 5 % (v/v) CO2 and used between passages 2 and 7. Cells were seeded out into appropriate plates or T75 flasks at specific densities to allow for 80-90 % confluence prior to incubation with inflammatory cytokines. On the day of the experiments, cells were placed in endothelial cell growth medium MV containing 1 % (v/v) FCS and stimulated with recombinant TNFα (Thermo Fisher Scientific, Paisley, UK) (10 ng/ml) or recombinant IL-1β (Millipore, Hertfordshire, UK) (10 ng/ml) for various times depending on whether TF mRNA, protein, activity or MV release was being measured.
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