Human il 6

Manufactured by BioLegend

Sourced in United States

Human IL-6 is a recombinant human cytokine. It is a glycoprotein that plays a central role in the host response to infection and tissue injury.

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Anti-human IL-6 ELISA MAX Standard Set Human IL-6 Human IL-6 ELISA kit Human IL-6 ELISA MAX Deluxe Kit APC anti-human IL-6 antibody ELISA MAXTM Deluxe Set Human IL-6 Human IL-6 ELISA MAX Deluxe LEGEND MAX Human IL-6 ELISA Kit ELISA MAX™ Deluxe Set Human IL-6 kit Recombinant human IL-6

11 protocols using human il 6

Quantitative Cytokine Profiling by ELISA

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The amounts of secreted IL-10 cytokine in human PBMC supernatants and IL-6 cytokine in TCM were measured by a sandwich enzyme-linked immunosorbent assay (ELISA) using human IL-10 (#430604, BioLegend) and human IL-6 (#430504, BioLegend) ELISA kits according to manufacturer’s instructions. Absorbance was read at 450 nm using Cytation 5 Cell Imaging Multi-Mode Reader (BioTek).

Martincuks A., Song J., Kohut A., Zhang C., Li Y.J., Zhao Q., Mak E., Rodriguez-Rodriguez L., Yu H, & Cristea M. (2021). PARP Inhibition Activates STAT3 in Both Tumor and Immune Cells Underlying Therapy Resistance and Immunosuppression In Ovarian Cancer. Frontiers in Oncology, 11, 724104.

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Cytokine Detection via Sandwich ELISA

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Cytokines were detected post-stimulation using a sandwich ELISA. All reagents were purchased from BioLegend: Human IL-6 (Capture: 501101, Detection: 501201, Standard: 570809), human IL-12p40 (Capture: 501702, Detection: 508801, Standard: 572109). ELISAs were performed as previously described (34 (link)).

Amici S.A., Young N.A., Narvaez-Miranda J., Jablonski K.A., Arcos J., Rosas L., Papenfuss T.L., Torrelles J.B., Jarjour W.N, & Guerau-de-Arellano M. (2018). CD38 Is Robustly Induced in Human Macrophages and Monocytes in Inflammatory Conditions. Frontiers in Immunology, 9, 1593.

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Conditional Retroviral Transduction of TCR

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Conditional retrovirus was produced using PLAT-E cells after cloning TCRα to a previously described construct (Morita et al., 2000 (link); Turner et al., 2010 (link)). TCR Vβ7 or Vβ8 transgenic Cd4-cre mice (usually also Tcra^−/−) were injected with 5-Fluorouracil (APP Pharmaceuticals) 3 days prior to bone marrow harvest. After harvest, bone marrow was cultured for 2 days in X-Vivo 10 (Lonza) supplemented with 15% FCS, 1% penicillin/streptomycin, mouse SCF, mouse IL-3, and human IL-6 (all from Biolegend). Stimulated cells were infected with retrovirus in the presence of polybrene (EMD Millipore) by spinfection. After 24 hr of additional culture, bone marrow cells were stained with antibodies against human CD4 or mouse Thy1.1, MACS-enriched (Miltenyi Biotec), and injected into lethally irradiated recipient mice. See also Supplemental Experimental Procedures.

McDonald B.D., Bunker J.J., Ishizuka I.E., Jabri B, & Bendelac A. (2014). Elevated T Cell Receptor Signaling Identifies a Thymic Precursor to the TCRαβ+CD4−CD8β− Intraepithelial Lymphocyte Lineage. Immunity, 41(2), 219-229.

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Monocyte Differentiation Assay

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Primary human monocytes, purchased from the Human Immunology Core at the University of Pennsylvania, were isolated from healthy volunteer donors following leukapheresis by negative selection. All specimens were collected under a University Institutional Review Board-approved protocol, and written informed consent was obtained from each donor. Isolated cells were incubated in RPMI-1640 medium supplemented with 5% heat-inactivated FBS. Cells were treated with 10 ng/ml human CSF-1, 100 ng/ml human IL-6 (BioLegend, 570808), and normal human ECs or patient tumor EC-CM.

Wang Q., He Z., Huang M., Liu T., Wang Y., Xu H., Duan H., Ma P., Zhang L., Zamvil S.S., Hidalgo J., Zhang Z., O’Rourke D.M., Dahmane N., Brem S., Mou Y., Gong Y, & Fan Y. (2018). Vascular niche IL-6 induces alternative macrophage activation in glioblastoma through HIF-2α. Nature Communications, 9, 559.

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Cytokine-Induced PD-L1 Expression in NSCLC

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Serum‐starved human NSCLC cell lines A549 and H1975 were treated with human transforming growth factor (TGF)‐β1 (2 ng/mL; R&D Systems) or human IL‐10 (10 ng/mL; R&D Systems), human tumor necrosis factor (TNF)‐α (10 ng/mL; BioLegend), human IL‐6 (10 ng/mL; BioLegend), human IL‐1α (10 ng/mL; R&D Systems) or human IL‐1β (10 ng/mL; R&D Systems) in serum‐free RPMI1640 containing 0.2% lactalbumin hydrolysate for 12 or 24 hours. Cells were also treated with vehicle alone as controls. The expression of PD‐L1 under treatment with these cytokines was analyzed by quantitative PCR.

Shima T., Shimoda M., Shigenobu T., Ohtsuka T., Nishimura T., Emoto K., Hayashi Y., Iwasaki T., Abe T., Asamura H, & Kanai Y. (2020). Infiltration of tumor‐associated macrophages is involved in tumor programmed death‐ligand 1 expression in early lung adenocarcinoma. Cancer Science, 111(2), 727-738.

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Quantifying Cytokine IL-6 Levels by ELISA

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IL-6 levels were measured by sandwich ELISA. Briefly, a 96-well
microtiter plate (Corning™ Clear Polystyrene flat-bottomed, 96-Well,
medium binding), was coated with 100 μL of capture antibody (4
μg/mL, anti -human IL-6, BioLegend) diluted in PBS and incubated overnight
at 4°C. After blocking with 1% BSA in PBS for 2 hours at room
temperature, samples were added and incubated overnight at 4°C. For
detection, plates were incubated with biotinylated detection antibody (1
μg/mL in blocking buffer, Biotin anti -human IL-6) for 2 hours at room
temperature, followed by addition of HRP-streptavidin (BioLegend) for an
additional 2 hours at room temperature. The reaction was visualized by the
addition of TMB. The reaction was stopped by addition of 2N sulfuric acid and
the absorbance was measured at 450 nm by plate reader. Wells were washed
thoroughly three times in PBS-Tween between every step. CRP was analyzed using
ELISA according to the manufacturer’s protocol (Enzo Life Sciences Inc.
Farmingdale, NY, USA).

Bach M., Moon J., Moore R., Pan T., Nelson J.L, & Lood C. (2019). A Neutrophil Activation Biomarker Panel in Prognosis and Monitoring of Patients with Rheumatoid Arthritis. Arthritis & rheumatology (Hoboken, N.J.), 72(1), 47-56.

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Murine and Human Cytokine Assay

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Secreted murine TNFα (Biolegend, San Diego, CA, USA), murine IL-6 (Biolegend), murine GM-CSF (Biolegend), human TNF (Biolegend) and human IL-6 (Biolegend) were measured by ELISA according to the manufacturer’s instructions.

Bénard A., Podolska M.J., Czubayko F., Kutschick I., Klösch B., Jacobsen A., Naschberger E., Brunner M., Krautz C., Trufa D.I., Sirbu H., Lang R., Grützmann R, & Weber G.F. (2022). Pleural Resident Macrophages and Pleural IRA B Cells Promote Efficient Immunity Against Pneumonia by Inducing Early Pleural Space Inflammation. Frontiers in Immunology, 13, 821480.

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Monocyte Isolation and Cytokine Treatment

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Primary human monocytes were provided by Human Immunology Core at the University of Pennsylvania. Peripheral blood mononuclear cells were collected from healthy human volunteer donors and monocytes were isolated following leukapheresis by negative selection. All specimens were collected under a University of Pennsylvania Institutional Review Board-approved protocol and written informed consent was obtained from each donor. We have complied with all relevant ethical regulations for work with human participants. Cells were incubated in RPMI-1640 medium supplemented with 5% fetal bovine serum (FBS) and treated with 10 ng/ml human CSF-1 (BioLegend, 574806) and 100 ng/ml human IL-6 (BioLegend, 570808).

Yang F., He Z., Duan H., Zhang D., Li J., Yang H., Dorsey J.F., Zou W., Nabavizadeh S.A., Bagley S.J., Abdullah K., Brem S., Zhang L., Xu X., Byrne K.T., Vonderheide R.H., Gong Y, & Fan Y. (2021). Synergistic immunotherapy of glioblastoma by dual targeting of IL-6 and CD40. Nature Communications, 12, 3424.

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Chemokine Profiles in Glial Cells

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Chemokine levels were measured in the culture supernatants of CRT-MG, U251-MG, and HMO6 in 60-mm culture plates at a final concentration of 9×10⁵ per well. After stabilization, cells were treated with either hydrogen peroxide (H₂O₂) or cobalt chloride (CoCl₂) for 24 h. For low and high glucose conditions, cells were cultured in low glucose DMEM (1,000 mg/L glucose), and high glucose DMEM (4,500 mg/L glucose), respectively. After treatment for 24 h, supernatants were collected, and IL-6, IL-8/CXCL8, and IP-10/CXCL10 concentrations were determined using a sandwich ELISA method with human IL-6 (BioLegend, San Diego, CA, USA), IL-8 (BioLegend), and IP-10 BD OptEIA™ (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer's instructions. The concentration of chemokines in each sample was determined by referencing a standard curve generated using known amounts of IL-6, IL-8, and IP-10.

Jung Y., Ahn S.H., Park S.H, & Choi Y.H. (2017). Effect of glucose level on chemical hypoxia- and hydrogen peroxide-induced chemokine expression in human glioblastoma cell lines. The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology, 21(5), 509-518.

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Quantification of IL-6 and IL-12p70 by ELISA

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Cell culture supernatants were collected 24h post-stimulation and were analyzed for the levels of IL-6 and IL-12p70, using the Biolegend human IL-6 and human IL-12p70 ELISA kits respectively, according to the manufacturer’s protocol. Absorbance was read at 450nm using a Synergy HTX multi-mode (BioTek) spectrophotometer within 15 minutes of stopping the reaction. Standard curves were generated and sample concentrations were calculated in pg/ml.

Gary E., O’Connor M., Chakhtoura M., Tardif V., Kumova O.K., Malherbe D.C., Sutton W.F., Haigwood N.L., Kutzler M.A, & Haddad E.K. (2020). Adenosine deaminase-1 enhances germinal center formation and functional antibody responses to HIV-1 Envelope DNA and protein vaccines. Vaccine, 38(22), 3821-3831.

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