The levels of TNF-α and IL-1β (Novus Biologicals, Inc.) in pleural effusions and the blood of mice were determined. Anti-CD8 antibody (Abcam), anti-PD1 antibody (EPR20665, Abcam), anti-PDL1 antibody (EPR20529, Abcam), anti-GAPDH antibody (ab8245, Abcam), and anti-β-actin antibody (13E5, CST) were used for antigen detection. Lymphocyte and tumor cell lines were co-cultured in 96-well plates at a ratio of 20:1. The LDH release experiment was then performed using the lactate dehydrogenase cytotoxicity test kit (Beyotime Biotechnology).
Il 1β
Manufactured by Novus Biologicals
Sourced in United States, United Kingdom
IL-1β is a cytokine that plays a key role in the inflammatory response. It functions as a pro-inflammatory agent, promoting the activation of immune cells and the production of other inflammatory mediators.
Automatically generated - may contain errors
Lab products found in correlation
Tnf α, by Abcam (3 mentions)
Ifn γ, by Thermo Fisher Scientific (2 mentions)
Protease and phosphatase inhibitor, by Roche (2 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Glial fibrillary acidic protein (gfap), by Thermo Fisher Scientific (2 mentions)
Icam 1, by Novus Biologicals (2 mentions)
Chemidoc imager, by Bio-Rad (2 mentions)
Tnf α, by Novus Biologicals (1 mentions)
Epr20665, by Abcam (1 mentions)
Lactate dehydrogenase cytotoxicity test kit, by Beyotime (1 mentions)
Ab8245, by Abcam (1 mentions)
Gapdh rabbit mab antibody, by Cell Signaling Technology (1 mentions)
Totallab software, by Bio-Rad (1 mentions)
Beclin 1, by Cell Signaling Technology (1 mentions)
Peroxidase conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Sqstm1 p62, by Cell Signaling Technology (1 mentions)
Tween 20, by Bio-Rad (1 mentions)
Blocking buffer, by Beyotime (1 mentions)
Gapdh, by Elabscience (1 mentions)
Lamp1, by Cell Signaling Technology (1 mentions)
12 protocols using il 1β
1
Cytokine Levels and Lymphocyte Cytotoxicity
2
Western Blot Analysis of Immune Markers
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Tissues or cultured BMMCs were lysed, and the protein was extracted. The protein lysate from each sample was separated electrophoretically on a sodium dodecyl sulfate-polyacrylamide gel and then transferred to a polyvinylidene fluoride (PVDF) membrane. After blocking with 5% nonfat milk in TBS-T (containing 0.1% Tween-20) for 2 hrs, the membranes were incubated with iNOS, P2X7, IL-1β (Novus Biologicals), and tryptase (AA1) antibodies in 5% nonfat milk in TBS-T overnight at 4°C. After washes with TBS-T, the membranes were incubated with the appropriate secondary antibodies for 2 hrs. The results were visualized using an ECL chemiluminescence system. GAPDH rabbit mAb antibody (Cell Signaling Technology) was also used as a probed control to ensure the loading of equivalent amounts of the sample proteins. The band densities were compared in TotalLab software (version 2.01; Bio-Rad, Hercules, CA).
3
Western Blot Analysis of Corneal Proteins
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Mice corneas (n = 6/group/time) were harvested at 3 days p.i. Protein extraction and concentration determination were performed as described previously.4 (link) The protein was separated on 12% acrylamide SDS-PAGE and transferred to polyvinylidene difluoride (PVDF; Solarbio) membrane. Membranes were washed in PBS containing 0.05% Tween-20 (Bio-Rad, Hercules, CA, USA) (PBST) three times. After being blocked with blocking buffer (Beyotime, Jiangsu, China) at 37°C for 2 hours, the membrane was incubated with antibodies to GAPDH (1:1000; Elabscience, Wuhan, China), CXCL-1 (1:1000; Affinity Biosciences, Jiangsu, China), LC3B (1:1000; Cell Signaling Technology, Danvers, MA, USA), SQSTM1/p62 (1:1000; Cell Signaling Technology), Beclin-1 (1:1000; Cell Signaling Technology), LAMP-1 (1:1000; Cell Signaling Technology), IL-1β (1:1000; NOVUS, Littleton, CO, USA), IL-18 (1:500; Abcam, Cambridge, MA, USA), HMGB1 (1:1000; Abcam), TNF-α (1:2000; Proteintech Group, Wuhan, China), or IL-10 (1:1000; Abcam) at 4°C overnight. Membranes were washed in PBS containing 0.05% Tween-20 (Bio-Rad, Hercules, CA, USA) (PBST) three times. Membranes were incubated with corresponding peroxidase-conjugated secondary antibodies (1:5000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 37°C for 1 hour. Membranes were developed by chemiluminescence (ECL; Thermo Fisher Scientific, Waltham, MA, USA).
4
Immunofluorescence Imaging of Kidney Inflammation
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After dehydration with 30% sucrose and embedding with optimal cutting temperature compound (OCT), the kidney tissue was sliced by a freezing microtome with a thickness of 7 μm. Next, kidney sections were incubated with rabbit or mouse antibodies against CD68 (1 : 200, lot: ab955, Abcam), NLRP3 (1 : 100, lot: F1716, Eterlife, UK), caspase-1 (1 : 200, lot: 00046167, Proteintech, USA), and IL-1β (1 : 200, lot: NB60-633, Novus, USA). Then, the sections were successively incubated with fluorescently labeled secondary antibodies and 4,6-diamidino-2-phenylindole (DAPI). Finally, the sections were observed by using fluorescence microscopy.
5
Flow Cytometric Analysis of Inflammatory Modulation of HLA Expression
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HeLa cells were seeded in a 12-well plate (1.2 X 105 cells/well) and treated with different concentrations of interleukin-1 beta (IL-1β) (Novus biologicals, CO, USA), IFN-γ (Peprotech, IL, USA) and tumor necrosis factor (TNF) (Origene, MD, USA) separately or in combination for 24h. Then, the cells were harvested, stained with monoclonal anti-HLA-A,B,C antibody (clone W6/32) conjugated with FITC (Cat. 311404, Biolegend, CA, USA) and analyzed by flow cytometry. IFN-γ at the concentration of 250 U/ml was chosen to establish inflammatory conditions (Supplementary Figure 1
6
Immunohistochemistry of Inflammatory Markers
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Joint tissues were incubated overnight at 4°C with primary antibodies against TNFα (Abcam, Cambridge, UK), IL-17 (Abcam), IL-6 (Abcam), and IL-1β (Novus Biologicals, Littleton, CO, USA). Subsequently, samples were incubated with a biotinylated streptavidin–peroxidase complex for 1 h, and the signals were developed using chromogen 3,3′-diaminobenzidine (Thermo Scientific, Rockford, IL, USA). The sections were examined under a photomicroscope (Olympus, Tokyo, Japan). The number of positive cells in high-power digital images (magnification, ×400) was counted using Adobe Photoshop software (Adobe, San Jose, CA, USA). Stained cells were counted independently by three observers, and the mean values were evaluated.
7
Quantification of Macrophage and Inflammatory Markers
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Macrophage and inflammation expression were evaluated by Immunohistochemical (IHC) analysis following a general IHC protocol. Tissue sections were immunostained at 4°C overnight with antibody against the macrophage marker RAM11 (DAKO, Santa Clara, CA, USA), or with antibodies against the inflammatory markers: TNF-α (ABCAM, Cambridge, United Kingdom), IL-1β (NOVUS, Littleton, CO, USA), or IL-6 (Santa Cruz Biotechnologies, CA, USA). The primary antibody was detected using a peroxidase-based kit (DAKO, Glostrup, Denmark), and visualized using 3,3′-diaminobenzidine substrate with enhancer (DAKO). The sections were subsequently counterstained with hematoxylin (DAKO). Digital images of the vessels were scanned using a SCN400 scanner (Leica), and histomorphometry was performed using LAS 4.2 software (Leica).
8
ELISA Analysis of Inflammatory Mediators
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Specific enzyme-linked immunosorbent assay (ELISA) kits for rats (Novus Biologicals, Centennial, CO, USA) were employed to assess the levels of inflammatory mediators in the hippocampal tissue, including tumor necrosis factor-α (TNF-α; catalog No.: NBP1-92681), interleukin-1β (IL-1β; catalog No.: NBP1-92,02), interleukin-6 (IL-6; catalog No.: NBP1-92697), interleukin-10 (IL-10; catalog No.: NBP1-92701), prostaglandin E2 (PGE2; catalog No.: NBP1-02321), and cyclooxygenase-2 (COX-2; catalog No.: NB600-971), according to the manufacturer’s guidelines.
9
Western Blot Analysis of Retinal Proteins
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Retinas were lysed in a RIPA buffer (EMD Merck, Kenilworth, USA) containing a phosphatase inhibitor PhosSTOP (Roche, Branchburg, USA). Protein was separated on NuPAGE gels (Life Technologies, Carlsbad, USA) and transferred to polyvinylidene difluoride membranes (Life Technologies). Blots were blocked with 5% nonfat milk in a TBST buffer (0.02 M Tris-base (pH 7.6), 0.8% NaCl, 0.1% Tween 20) for 60 min. After rinsing with the TBST buffer, samples were incubated with primary antibodies at 4 °C overnight. The antibodies we used were as follows: iNOS (1:500, Abcam, Cambridge, UK), Cl-caspase-3 (1:1000, Cell Signaling, Danvers, USA), IL-1β (1:250, Novus Biologicals, Littleton, USA), TNF-α (1:500, Abcam), CNTF (1:3000, Abcam), p-ERK (1:1000, Cell Signaling), and GAPDH (1:5000, Cell Signaling). Membranes were washed twice with the TBST buffer, followed by incubation with appropriate Horseradish Peroxidase (HRP)-conjugated secondary antibodies (1:10000, Jackson Immunoresearch, PA, USA) at room temperature for 1 hour and detected with Immobilon® ECL Ultra Western HRP Substrate (Millipore, Billerica, USA). Results were quantified using ImageJ software (n = 6 in each group).
10
Quantitative Western Blot Analysis
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