The cells were pretreated with SSME (25, 50, 100, and 200 µg/mL) for 2 h and stimulated with LPS (1 µg/mL) for 24 h. After stimulation, the levels of each cytokine were measured in the cell culture supernatants. The purified anti-IL-6 (1:250; #554400), anti-IL-10 (1:250; #551215) (BD Bioscience), anti-IL-1β (1:200; #14-7012-85), and anti-TNF-α (1:250; #14-7423-85) (Thermo Fisher Scientific Inc.) antibodies were coated onto 96-well plates and incubated overnight at 4 °C. Next, the plates were washed with PBS containing 0.05% Tween 20 and incubated with PBS containing 1% bovine serum albumin (BSA) at RT for 1 h. After that, the plates were washed, and the supernatant samples were added for 2 h. The plates were washed, and the detection antibodies were added for 1 h. Next, streptavidin-conjugated alkaline phosphatase (1:250; AKP, #554065; BD Bioscience) was added after washing. Substrate buffer [10% diethanolamine (#3032-4400), 0.1% MgCl2·6H2O (#5503-44), 0.2% NaN3 (#7530-4105) (Daejung Chemicals & metals Co., Siheung, Republic of Korea) and 4-nitrophenyl phosphate (#N2765, Sigma-Aldrich)] was added to each well. The absorbance of the samples was measured at 405 nm using a microplate reader.
Anti il 6
Manufactured by BD
Sourced in United Kingdom, United States
Anti-IL-6 is a laboratory reagent that binds and neutralizes the cytokine Interleukin-6 (IL-6). It is used in research applications to study the role of IL-6 in various biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Anti il 10, by BD (2 mentions)
Anti tnf α, by Thermo Fisher Scientific (1 mentions)
Anti il 1β, by Thermo Fisher Scientific (1 mentions)
4 nitrophenyl phosphate, by Merck Group (1 mentions)
Facsaria, by BD (1 mentions)
Soluble anti cd28, by BD (1 mentions)
Interleukin 6 (il 6), by Miltenyi Biotec (1 mentions)
Positive selection, by Miltenyi Biotec (1 mentions)
Cgp37157, by Bio-Techne (1 mentions)
Anti cd3 2c11, by BD (1 mentions)
Fk506, by InvivoGen (1 mentions)
Anti fibronectin, by BD (1 mentions)
Anti α sma, by Abcam (1 mentions)
Anti ifn γ, by BD (1 mentions)
Anti il 17, by R&D Systems (1 mentions)
Anti il 1β, by BD (1 mentions)
Anti il 8, by BD (1 mentions)
Anti tgf β, by BD (1 mentions)
Anti il 4, by BD (1 mentions)
Anti tnf α, by BD (1 mentions)
7 protocols using anti il 6
1
Cytokine Measurement in LPS-Stimulated Cells
2
Isolation and Activation of CD4 T Cells
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CD4 cells were isolated from spleen and lymph nodes by negative selection as previously described (Diehl et al., 2002 (link)). For Stat3+/+ and Stat3−/− mice, CD4 cells were purified by cell sorting (FACS-Aria; Becton Dickinson). CD4 cells were activated with plate-bound anti-CD3 (2C11) (5 μg/ml) and soluble anti-CD28 (1 μg/ml) (BD Pharmingen, San Diego, CA) mAbs in the presence or absence of IL-6 (50 ng/mL) (Miltenyi Biotec, Auburn, CA). Pharmacological inhibitors were added to culture 42 hr after activation and supernatants were harvested 6 hr later. APCs were purified by depleting CD4 and CD8 T cells using positive selection (Miltenyi), and followed by irradiation treatment (2000 rad). APCs and OT-II CD4 cells were co-cultured at 4:1 ratio in the presence of 5 μM OVA323-339 peptide (Barnden et al., 1998 (link)) with or without IL-6 (50 ng /mL) (Miltenyi) or anti-IL-6 (2.5 μg /mL) (BD Pharmingen).
Pharmacological inhibitors used were CGP-37157 (Tocris Bioscience, Ellisville, MO) (10 μM), CCCP (2 μM), rotenone (2 μM), antimycin (2 μM), Ru360 (10 μM), FK506 (InvivoGen, San Diego, CA) (10 nM), Stattic (10 μM).
Pharmacological inhibitors used were CGP-37157 (Tocris Bioscience, Ellisville, MO) (10 μM), CCCP (2 μM), rotenone (2 μM), antimycin (2 μM), Ru360 (10 μM), FK506 (InvivoGen, San Diego, CA) (10 nM), Stattic (10 μM).
3
Immunohistochemical Evaluation of IL-6, Fibronectin, and α-SMA
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Immunohistochemical staining assays were performed to evaluate the expression and distribution of IL-6, fibronectin, α-SMA. Immunohistochemistry staining was performed as described (7 (link)). Anti-α-SMA (1:100; Abcam, UK), Anti-IL-6 (1:500; BD, USA), Anti-fibronectin (1:500; BD, USA) antibodies were used as primary antibodies.
4
Cytokine Profiling in Blood Samples
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Blood samples were drawn at three different moments: at baseline, after 6 months of treatment (T6), and at the end of the follow-up (T12). SPT was carried out at baseline and the end of the follow-up (T12).
For cytokines analysis, peripheral blood was collected and centrifuged at 2,500 rpm for 10 min at 4°C. The serum samples were stored and frozen at −80°C until all analysis. Briefly, wells in a 96-well plate were covered and incubated with purified antibodies anti-IL-17 (R&D System, Minneapolis, MN, EUA), anti-IFN-γ, anti-IL-1β, anti-IL-4, anti-IL-6, anti-IL-8, anti-IL-10, anti-TNF-α, or anti-TGF-β (BD Bioscience, San Diego, CA, EUA), and enzyme-linked immune sorbent assays (ELISA) were carried out, according to the manufacturer's instructions.
For cytokines analysis, peripheral blood was collected and centrifuged at 2,500 rpm for 10 min at 4°C. The serum samples were stored and frozen at −80°C until all analysis. Briefly, wells in a 96-well plate were covered and incubated with purified antibodies anti-IL-17 (R&D System, Minneapolis, MN, EUA), anti-IFN-γ, anti-IL-1β, anti-IL-4, anti-IL-6, anti-IL-8, anti-IL-10, anti-TNF-α, or anti-TGF-β (BD Bioscience, San Diego, CA, EUA), and enzyme-linked immune sorbent assays (ELISA) were carried out, according to the manufacturer's instructions.
5
Monocyte-derived Dendritic Cells Interaction with Bregs
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MDDCs were generated by culturing monocytes from healthy donors in cRPMI supplemented with 100 ng/mL GM-CSF (PeproTech) and 50 ng/mL IL-4 (PeproTech)58 (link). MDDCs were treated with LPS (100 ng/mL; Invitrogen). LPS before coculturing them with B cells. FACS-sorted TIGIT+ Bregs or TIGIT− B cells were cocultured with MDDCs at 1:1 ratio for 2 days. Cell stimulation cocktail containing PIBM was added 5 h prior to harvest. Cells were washed, human Fc-blocker was added, and live–dead staining was performed. For surface staining, cells were labeled with anti-CD40 (5C3), anti-CD19 (SJ25C1), anti-CD11c (B-ly6), anti-CD80 (L307.4), anti-CD83(HB15e), anti-CD86 (IT2.2), anti-HLA-DR (L243), anti-CCR7 (150503), and anti-ICOS-L (2D3). Cells were then washed, fixed, and permeabilized using Cytofix/Cytoperm (BD Biosciences), followed by staining them with anti-IL-10 (JES3-9D7), anti-IL-6 (MQ2-13A5), and anti-IL-12A (2Y37).
6
Isolation and Activation of CD4+ T Cells
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PBMCs were isolated as described before and erythrocytes were depleted. Next, CD4+ T cells were isolated with magnetic beads according to the manufacturer’s instructions (Miltenyi Biotec). CD4+ T cells were incubated in RPMI 1640 Glutamax (Gibco) supplemented with 100 µM β-mercaptoethanol (Life Technologies) for 60 min 37°C and then stained extracellularly for CD4. Afterwards, 1×106 cells were taken up in 500 µL prewarmed RPMI 1640 Glutamax (Gibco), supplemented with 100 U/mL penicillin/streptavidin (Gibco) and 10% FCS (Sigma) and incubated either with or without 20 ng/mL IL-23, 20 µg/mL anti-IL6 (BD Biosciences) and 2 µg/mL anti-IL22 (R&D) for 5 min at 37°C. Stimulation was stopped and cells were fixed by addition of cold 4% paraformaldehyde in phosphate buffered saline (PBS) and incubated for 10 min at room temperature. After a single wash with PBS, cells were permeabilised in 70% ice-cold methanol in PBS for 30 min on ice. The cells were then stained intracellularly for 30 min at room temperature with an antibody specific for phosphorylated STAT3 (pSTAT3) (BD Biosciences no. 557815) and analysed by flow cytometry.
7
Induction of Experimental Autoimmune Encephalomyelitis
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Donor C57BL/6 mice were subcutaneously immunized with 150 μg MOG35–55 peptide (Genemed Synthesis Inc., San Antonio, TX, USA) emulsified in Complete Freund’s adjuvant (5 mg/mL heat-killed M. tuberculosis). This was followed by an intraperitoneal (I.P.) injection of 250 ng of Bordetella pertussis toxin (List Biological Laboratories Inc., Campbell, CA, USA) in PBS at day 0 and day 2 post-immunization. Donor mice were sacrificed day 10 post-immunization, and spleens and lymph nodes were harvested and mechanically disrupted to obtain a single-cell suspension. 2.5 × 106 cells/mL were stimulated for three days with MOG35–55 (10 µg/mL), IL-23 (10 ng/mL; R & D Systems, Minneapolis, MN, USA) and anti-IFN-γ (10 µg/mL; eBioscience, San Diego, CA, USA) in the presence or absence of IFN-β (100 U/mL; PBL, Novato, CA, USA) in complete RPMI 1640 (Gibco, Waltham, MA, USA). To block IL-6 signaling, cells were cultured with anti-IL-6 (10 μg/mL; BD Biosciences, Franklin Lakes, NJ, USA).
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