U2OS and MG-63 osteosarcoma cells and human normal osteoblastic hFOB 1.19 cells were obtained from the Shanghai Institute of Biochemistry and Cell Biology (Chinese Academy of Sciences, Shanghai, China). The human glioblastoma cell line, T98G, was purchased from American Type Culture Collection (Rockville, MD, USA). Dulbecco's Modified Eagle Medium (DMEM) cell culture medium and fetal bovine serum (FBS) were purchased from Hangzhou Sijiqing Biological Engineering Material Co. Ltd. (Hangzhou, Zhejiang, China). Antibodies against OPN were purchased from Abcam (Cambridge, MA). N-acetyl cysteine and recombinant human IL-6 were purchased from Sigma (St. Louis, MO, USA). Enhanced chemiluminescence (ECL) western blotting kits were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).
Recombinant human il 6
Manufactured by Merck Group
Sourced in United States, Italy
Recombinant human IL-6 is a cytokine produced using recombinant DNA technology. It is a protein that plays a role in immune and inflammatory responses.
Automatically generated - may contain errors
Lab products found in correlation
Hepg2, by American Type Culture Collection (3 mentions)
Hydroxyurea, by Merck Group (2 mentions)
Nutlin 3, by Merck Group (2 mentions)
Cycloheximide (chx), by Merck Group (2 mentions)
Ncm460 cell line, by INCELL (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Anti β catenin, by Santa Cruz Biotechnology (2 mentions)
Anti il 6, by Santa Cruz Biotechnology (2 mentions)
Anti stat3, by Cell Signaling Technology (2 mentions)
Anti bcl xl, by Cell Signaling Technology (2 mentions)
Anti β actin, by Merck Group (2 mentions)
Antibodies against opn, by Abcam (1 mentions)
N acetylcysteine, by Merck Group (1 mentions)
Tgf β, by Merck Group (1 mentions)
Anti il 6 antibody, by R&D Systems (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Bel 7402, by American Type Culture Collection (1 mentions)
Bel 7404, by American Type Culture Collection (1 mentions)
Anti il 6, by Merck Group (1 mentions)
Anti e cadherin clone 32a8, by Cell Signaling Technology (1 mentions)
13 protocols using recombinant human il 6
1
Osteosarcoma and Glioblastoma Cell Lines
2
Modulating Cell Signaling Pathways
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Recombinant human IL-6 and EGF were purchased from Sigma-Aldrich (H7416 and E9644), reconstituted in phosphate-buffered saline, and used at final concentrations of 25 and 50 ng/mL, respectively. TGFβ was purchased from Sigma-Aldrich and used at a final concentration of 5 ng/mL. The MEK1/2 inhibitor PD98059 and STAT3 inhibitor S3I-201 were purchased from Calbiochem, dissolved in dimethyl sulfoxide (DMSO), and used at final concentrations of 50 μM and 100 μM, respectively. Cells were preincubated with the inhibitors for 1 h at 37°C and then incubated in the absence or presence of IL-6. The experiments ran for 4 days and media with inhibitor was changed every 2 days. BIX15 was purchased from Vibrant Pharma (Brantford, ON), dissolved in DMSO, and used at a final concentration of 5 μM. Control cells were treated with the same amount of DMSO (Vehicle).
3
Exploring Liver Cancer Cell Responses
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Bel-7404, Bel-7402, and HepG2 cells were obtained from American Type Culture
Collection (ATCC, Manassas, VA, U.S.A.) and respectively cultured in RPMI 1640
and DMEM (Invitrogen Life Technologies) with 10% FBS (Invitrogen Life
Technologies) at 37°C in a humidified atmosphere with 5%
CO2.
On reaching 60–80% confluence, both cell lines were treated with
BMSC-CM, serum-free DMEM, 2% FBS-containing DMEM, recombinant human IL-6
(Sigma–Aldrich Corp., St. Louis, MO, U.S.A.) or the anti-IL-6 antibody
(R&D Systems, Inc., Minneapolis, MN, U.S.A.).
Collection (ATCC, Manassas, VA, U.S.A.) and respectively cultured in RPMI 1640
and DMEM (Invitrogen Life Technologies) with 10% FBS (Invitrogen Life
Technologies) at 37°C in a humidified atmosphere with 5%
CO2.
On reaching 60–80% confluence, both cell lines were treated with
BMSC-CM, serum-free DMEM, 2% FBS-containing DMEM, recombinant human IL-6
(Sigma–Aldrich Corp., St. Louis, MO, U.S.A.) or the anti-IL-6 antibody
(R&D Systems, Inc., Minneapolis, MN, U.S.A.).
4
Cytokine ELISA and Cell Signaling Assays
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Cytokine enzyme-linked immunosorbent assay (ELISA) kit (Genzyme, Cambridge, MA, USA), recombinant human IL-6 at a final concentration of 50 ng/ml (Sigma-Aldrich, Milan, Italy), proteasome inhibitor MG-132 at a final concentration of 10 mM (Calbiochem; Merck Chemicals Ltd., Nottingham, UK), cycloheximide at a final concentration of 20 µg/ml (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), hydroxyurea (Sigma-Aldrich; Merck KGaA), 5-FU (Fluorouracil; Teva Pharma Italia, Milan, Italy), Nutlin-3 (Sigma-Aldrich; Merck KGaA), anti-E-cadherin (clone 32A8, Cell Signaling Technology, Inc., Danvers, MA, USA), anti-IL-6 (Sigma-Aldrich; Merck KGaA), anti-p53 (Novocastra, Laboratories, Ltd., Newcastle upon Tyne, UK), and p53 Taqman gene expression quantitative assay kit (Applied Biosystems, Foster City, CA, USA) were used in the present study.
5
Molecular Response of p53 Wild-Type and Deficient Cell Lines
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HepG2, SW1990 and LS174T cell lines were obtained from American Type Culture Collection, and the NCM460 cell line was purchased from INCELL Corporation (San Antonio, TX, USA). All the cell lines were p53 wild-type. The HCT116 p53−/− cell line was a generous gift from Professor Bert Vogelstein.
The recombinant human IL-6 (Sigma-Aldrich, Milan, Italy) was used at a final concentration of 50 ng/ml; the proteasome inhibitor MG-132 (Calbiochem, Merck, Nottingham, UK) was used at a final concentration of 10 mM ; cycloheximide (Sigma-Aldrich) was used at a concentration of 20 μg/ml; hydroxyurea (Sigma-Aldrich) was used at 3.4 mM ; 5-FU (Fluorouracile, Teva Pharma Italia, Milan, Italy) was used at a dose of 20 μg/ml; Nutlin-3 (Sigma-Aldrich) was used at a concentration of 5 mM .
The recombinant human IL-6 (Sigma-Aldrich, Milan, Italy) was used at a final concentration of 50 ng/ml; the proteasome inhibitor MG-132 (Calbiochem, Merck, Nottingham, UK) was used at a final concentration of 10 m
6
BRD and Notch Pathway Protein Analysis
7
Anti-Inflammatory Drugs and Cell Lines
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HepG2 and MCF10A were obtained from ATCC between 2005 and 2009. The NCM460 cell line was purchased from INCELL Corporation (San Antonio, TX, USA) in 2014. All the cell lines were p53 wild-type. C57BL/6JOlaHsd mice were used for primary mouse embryonic fibroblast (MEF) isolation. Recombinant human IL-6 (Sigma-Aldrich, Milan, Italy) was used at a final concentration of 50 ng/ml; mouse IL-6 (Sigma-Aldrich) was used at a final concentration of 40 μg/kg; aspirin (acetylsalicylic acid, Sigma-Aldrich) was used at 0.1, 0.3, 0.5, 1, 1.5 and 3 mM in cell lines, at 30 mg/kg in mice; salicylate (Sigma-Aldrich) was used at 0.5 mM; mesalazine (5-ASA, Sigma-Aldrich) was used at 1 mM.
8
Investigating STAT3 Signaling in IL-6 Receptor Regulation
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Recombinant human sIL-6R and goat anti-IL-6 were purchased from R&D Systems. The mouse anti-STAT3, rabbit anti-phosphor-STAT3 (Tyr705) (pSTAT3), and horseradish peroxidase (HRP)-linked anti-rabbit and anti-mouse antibodies were from Cell Signaling Technology, and recombinant human IL-6 and mouse anti-IL-6R (B-R6) were from Millipore. Rabbit anti-IL-6R (C-20) was obtained from Santa Cruz Biotechnology, mouse anti-cd49b was from BD Biosciences, mouse anti-LAMP-1 (H4A3) was from the Developmental Studies Hybridoma Bank, and NT was from Sigma. The murine monoclonal anti-SorLA (31 (link)) and the rabbit polyclonal anti-SorLA have previously been described (32 (link)). Secondary antibodies conjugated with Alexa Fluor dyes were all from Invitrogen.
9
Antibody Immunoblotting Protocol
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The following antibodies were used in this study: anti-SULF2, anti-IL-6, and anti-β-catenin from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-Bcl-XL, anti-STAT3, and anti-phospho-STAT3 from Cell signaling Technology (Danvers, MA, USA); anti-β-actin from Sigma-Aldrich (St. Louis, MO, USA). Recombinant human IL-6 was purchased from Millipore (Bedford, MA, USA).
10
Antibody-Based Protein Analysis
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The following antibodies were used in the present study: anti-SOD2 from Enzo Life Sciences (Farmingdale, NY, USA); anti-IL-6 and anti-β-catenin from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-Bcl-XL, anti-STAT3, anti-Src, and anti-phospho-Src from Cell Signaling Technology (Danvers, MA, USA); and anti-β-actin from Sigma-Aldrich (St. Louis, MO, USA). Recombinant human IL-6 was purchased from Millipore (Darmstadt, Germany).
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