MEFs were lysed in 1% Triton-X100 lysis buffer [20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton-X100, 1.25 mM NaF, 0.2 mM NaVO3, 2 mM β-glycerophosphate, 10 mM N-ethylmaleimide and complete TM protease inhibitor cocktail (Roche)], each lysate was normalized by BCA protein assay (Thermo). IKKγ immunoprecipitations were performed using a sheep polyclonal anti-IKKγ antibody at 4 °C for 4 h. Protein G Dynabeads (Thermo) were added for the final 1 h of incubation. Active TNFR complex IPs were performed with magnetic anti-Flag (M2) beads (Sigma) by incubation at 4 °C for 2 h. IL-1β IPs were performed by incubating lysates with either biotinylated anti-IL-1β (BioLegend B122) or biotinylated IgG1 isotype control for 16 h followed by adding Neutravidin Agarose (Pierce) for 1 h. Beads were then washed three times and samples were eluted from the beads using 2X LDS buffer (Thermo) containing 715 mM β-mercaptoethanol.
Anti il 1β
Manufactured by BioLegend
Sourced in China, United States
Anti-IL-1β is a lab equipment product that can be used to detect and measure levels of the cytokine Interleukin-1 beta (IL-1β) in biological samples. It is an antibody-based reagent designed for use in various immunoassay techniques.
Automatically generated - may contain errors
Lab products found in correlation
Anti il 6, by BioLegend (5 mentions)
Anti tnf α, by BioLegend (4 mentions)
Bca protein assay, by Thermo Fisher Scientific (4 mentions)
Protein g dynabead, by Thermo Fisher Scientific (2 mentions)
Magnetic anti flag m2 beads, by Merck Group (2 mentions)
Lds buffer, by Thermo Fisher Scientific (2 mentions)
Complete protease inhibitor cocktail, by Roche (2 mentions)
Mouse il 10 duoset elisa kit, by R&D Systems (2 mentions)
Biotinylated detection mabs, by BioLegend (2 mentions)
Hrp conjugated avidin d, by Vector Laboratories (2 mentions)
Oil red o, by Merck Group (1 mentions)
Anti il 8, by R&D Systems (1 mentions)
Interleukin 6 (il 6), by BioLegend (1 mentions)
Il 1β, by BioLegend (1 mentions)
Tnf α, by Miltenyi Biotec (1 mentions)
Anti cd8a, by BioLegend (1 mentions)
Anti cd4, by BioLegend (1 mentions)
8 protocols using anti il 1β
1
Immunoprecipitation of Inflammatory Complexes
2
Skin-Derived Adipogenic Signaling
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Skin culture was prepared by taking 1 cm2 skin sections from 6-months-old normal or KCASP1Tg mice, minced with scissors, and harvested in 2 mL of RPMI 1640 containing 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin plated in a 24-well culture plate (Coster, NY, USA). In some experiments the skin post culture medium was treated with 1 µg of anti-IL-1α, anti-IL-1β, or a mixture of anti IL-1α and β neutralizing antibodies (BioLegend), and then incubated for 2 hours. The mouse embryonic fibroblast adipose-like cell line 3T3-L1 (ATCC, Manassas, VA) was cultured as previously reported for 10 days [15] (link), and then differentiated into mature adipocytes. The medium was changed using the following supplementation: skin culture supernatant derived from normal mice, KCASP1Tg, anti-IL-1α and/or anti-IL-1β neutralizing antibodies-treated KCASP1Tg skin supernatant. On day 14, cultured cells were rinsed with PBS, stained with oil red O (Sigma-Aldrich, St. Louis, MO) and haematoxylin, and then observed under microscope (n = 7, each group).
3
Recombinant MIF and Antagonist Preparation
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Recombinant human MIF (rhMIF) was prepared as described elsewhere (46 (link)) (endotoxin content < 0.1 EU/ml). MIF antagonist MIF098 [3-(3-hydroxybenzyl)-5-methylbenzooxazol-2-one] was dissolved in DMSO at a concentration of 149 µM (47 (link)). The neutralizing anti-MIF monoclonal antibody (clone NIHlllD.9) was obtained from ascites after purification using protein A/G spin column and resuspended at 5.15 mg/ml (48 (link), 49 (link)). A CD74 blocking antibody (BD Pharmingen, clone LN2), the recombinant human cytokines IL-6, IL-8, IL-1β (BioLegend), and TNFα (MiltenyiBiotec), and the cytokine neutralizing antibodies anti-IL-8 (R&D Systems), anti-IL-6, anti-IL-1β, and anti-TNFα (BioLegend) were obtained.
4
Quantification of Cytokine Levels in Cell Supernatants
5
Immunomodulation of Tumor Growth
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Six-week-old male C57BL/6 mice were intraperitoneally injected with sh-Agpat4- or sh-NC-transfected MC-38 cells (4.0 × 106 cells in 0.1 ml of PBS per mouse). The tumor-bearing mice were treated with neutralizing antibodies, including anti-CD4 (#100401, BioLegend, China), anti-CD8a (#100775, BioLegend, China), anti-IL1-β (#503502, BioLegend, China), anti-IL-6 (#504506, BioLegend, China), and anti-TNFα (#506325, BioLegend, China) or IgG (#400413, BioLegend) as a control.
6
Recombinant Human MIF and Antagonist Preparation
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Recombinant human MIF (rhMIF) was prepared as described elsewhere [24 (link)] (endotoxin content < 0.1 EU/mL). MIF antagonist MIF098 [3-(3-hydroxybenzyl)-5-methylbenzooxazol-2-one] was dissolved in DMSO at a concentration of 149 µM [25 (link)]. The neutralizing anti-MIF monoclonal antibody (clone NIHlllD.9) was obtained from ascites after purification using protein A/G spin column and resuspended at 5.15 mg/mL [26 (link),27 (link)]. The cytokine neutralizing antibodies anti-IL-6 and anti-IL-1β (BioLegend, San Diego, CA, USA) were obtained.
7
Immunoprecipitation of Inflammatory Complexes
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MEFs were lysed in 1% Triton-X100 lysis buffer [20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton-X100, 1.25 mM NaF, 0.2 mM NaVO3, 2 mM β-glycerophosphate, 10 mM N-ethylmaleimide and complete TM protease inhibitor cocktail (Roche)], each lysate was normalized by BCA protein assay (Thermo). IKKγ immunoprecipitations were performed using a sheep polyclonal anti-IKKγ antibody at 4 °C for 4 h. Protein G Dynabeads (Thermo) were added for the final 1 h of incubation. Active TNFR complex IPs were performed with magnetic anti-Flag (M2) beads (Sigma) by incubation at 4 °C for 2 h. IL-1β IPs were performed by incubating lysates with either biotinylated anti-IL-1β (BioLegend B122) or biotinylated IgG1 isotype control for 16 h followed by adding Neutravidin Agarose (Pierce) for 1 h. Beads were then washed three times and samples were eluted from the beads using 2X LDS buffer (Thermo) containing 715 mM β-mercaptoethanol.
8
Cytokine Detection in Cell Cultures
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For the detection of cytokines in the cell culture supernatants or sera, ELISAs were performed as described previously (17) , using the primary capture mAbs anti-TNFα, anti-IL-6, and anti-IL-1β and their corresponding biotinylated detection mAbs (Biolegend, San Diego, CA). Other ELISA reagents included: recombinant mouse TNFα, IL-6, and IL-1β as standards (Biolegend, San Diego, CA), HRP-conjugated avidin D (Vector Laboratories, Burlingame, CA), and TMB microwell peroxidase substrate and stop solution (Kirkegaard and Perry Laboratories, Gaithersburg, MD). IL-10 cytokine was measured using Mouse IL-10 DuoSet ELISA kit (R&D Systems, Minneapolis, MN) according to manufacturer's instructions. Biological replicates were used, representing independent BMDM cultures from individual mice.
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