Supernatant from mock- or C. pneumoniae-infected monocytes was collected, supplemented with the recommended 1× concentration of Halt protease inhibitor cocktail (Thermo Scientific, Rockford, IL) and stored at −80°C for further analysis. For intracellular ELISA, the flasks/wells were incubated on ice for 5 min to detach the adherent cells, gently scraped, separated by centrifugation (10 min, 300×g) and counted. The cells were lysed for intracellular ELISA using RIPA buffer (Thermo Scientific, Rockford, IL) with 1× concentration of Halt protease inhibitor cocktail (Thermo Scientific).The cell lysate was collected by spinning down at 10,000 rpm at 4°C for 10 min and stored at −80°C for further analysis. The supernatant or cell lysates were assayed for IL-1β using Human IL-1β ELISA Set II (BD Biosciences, San Diego, CA) according to manufacturer’s instructions.
Human il 1β elisa set 2
Manufactured by BD
Sourced in United States
The Human IL-1β ELISA Set II is a laboratory equipment designed for the quantitative measurement of interleukin-1 beta (IL-1β) in human biological samples. It utilizes the enzyme-linked immunosorbent assay (ELISA) technique to detect and quantify the target analyte.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Thermo Fisher Scientific (1 mentions)
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Thioflavin t tht, by Merck Group (1 mentions)
Mcc950 pz0280, by Merck Group (1 mentions)
Human tnf α duoset elisa, by R&D Systems (1 mentions)
Easysep human monocyte isolation kit, by STEMCELL (1 mentions)
Succinate, by Merck Group (1 mentions)
Mouse il 1β elisa set, by BD (1 mentions)
Mouse il 1β, by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharides (lps), by InvivoGen (1 mentions)
Human total il 18 duoset elisa, by R&D Systems (1 mentions)
Ldh activity assay, by Promega (1 mentions)
Dulbecco s phosphate buffered saline dpbs, by Biowest (1 mentions)
Ch 2 cl 2, by Scharlab (1 mentions)
Ch3cn, by Scharlab (1 mentions)
Pluronic f 127, by Merck Group (1 mentions)
Crystal violet, by Merck Group (1 mentions)
Cell proliferation reagent wst 1, by Merck Group (1 mentions)
Penicillin streptomycin, by Biowest (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
10 protocols using human il 1β elisa set 2
1
Intracellular IL-1β ELISA in C. pneumoniae-infected Monocytes
2
Amyloid-beta Aggregation and Inflammation Assays
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Aβ42 was purchased form the Peptide Institute (Ibaraki, Osaka, Japan). Anti-Aβ mouse monoclonal antibody (6E10) and anti-mouse immunoglobulin G (IgG) were purchased from BioLegend (San Diego, CA, USA) and R&D Systems (Minneapolis, MN, USA), respectively. Thioflavin T (ThT) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Anti-FLAG monoclonal antibody M2 (F1804) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Human IL-1β ELISA Set II was purchased from BD Biosciences (San Jose, CA, USA). Anti-cleaved caspase-1 rabbit monoclonal antibody (Asp297) (D57A2) (S4199) was purchased from Cell Signaling Technology (Danvers, MA, USA). MCC950 (PZ0280) was purchased from Sigma-Aldrich (St. Louis, MO). Isoliquiritigenin (I0822) was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan).
3
Quantification of IL-1β Secretion
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Human IL-1β ELISA Set II (BD Bioscience) was used to detect secreted IL-1β in cell culture supernatants after stimulation of monocytes for 16 h or THP-1 cells for 8 h at cell culture conditions (37 °C/5% CO2). ELISA was performed as described in manufacturer’s instructions.
4
Profiling Cytokine Secretion in Systemic Sclerosis Monocytes
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PBMCs were obtained from a patient with SSc. Monocytes were negatively purified using an EasySep Human Monocyte Isolation Kit (STEMCELL Technologies #19359). The CEACAM+ monocyte subset was isolated using CEACAM MicroBeads (Miltenyi). The cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin and stimulated with 50 ng/mL LPS in a 96-well round-bottom plate at a concentration of 1 × 106 cells/mL for 6 h. Cytokine levels in the culture medium supernatant were measured using the Human TNF-α Duoset ELISA (R&D Systems) and Human IL-1β ELISA Set II (BD Biosciences) following the manufacturer’s instructions.
5
Macrophage Activation and Cytokine Assay
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U937 or mouse macrophages derived from 7–9-wk-old age-matched animals were incubated for up to 24 h in 96-well flat-bottom plates at a density of 105 cells per well in RPMI-1640 containing 1 or 100 ng/ml LPS (InvivoGen), 500 µM succinate (sodium succinate dibasic hexahydrate; Sigma-Aldrich), 10 ng/ml mouse IL-1β (PeproTech), 180 µg/ml MSU (Enzo Life Sciences), 5 µM GPR91 antagonist GPR91A1 (synthesized in house from Bhuniya et al., 2011 (link)), and 10% human RA SFs (Asterand). After 24 h (or 7 h in the case of culture with 1 ng/ml LPS [2 h] followed by 180 µg/ml MSU [5 h]), cells were collected and processed for quantitative PCR as described in the next section. Cytokines in supernatants were analyzed by ELISA using the mouse IL-1β ELISA Set or human IL-1β ELISA Set II (both from BD) according to the manufacturer’s instructions.
6
Nigericin-induced IL-1β Cytokine Assay
7
Quantifying Cytokine Levels in Cell Culture
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Concentrations of cytokines in cell culture supernatants were determined by ELISA according to the manufacturer's instructions. The following kits were used: Human IL‐1β ELISA Set II (BD Biosciences), Human Total IL‐18 DuoSet ELISA (R&D Systems), Human Interferon‐gamma DuoSet ELISA (R&D Systems), and Human IL‐4 DuoSet ELISA (R&D Systems).
8
Synthesis and Characterization of Hexanuclear Molybdenum Clusters
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The chemical reagents tetraethylorthosilicate (TEOS), n-cetyltrimethylammonium bromide (CTABr), sodium hydroxide (NaOH), (3aminopropyl) triethoxysilane (APTES, 99 %), Pluronic® F-127, 9,10anthracenediyl-bis(methylene)dimalonic acid (ABDA, ≥90 %), as well as the reagents to prepare the phosphate-buffered saline (PBS) solution namely, RPMI 1460 media (R8758), lipopolysaccharide (LPS) from Escherichia coli O111:B4 (L2630-100MG), nigericin (NG) (N7143-5MG), Crystal Violet, Cell Proliferation Reagent WST-1, NaCl (≥99 %), KCl (≥99.0 %), Na 2 HPO 4 (≥99.0 %) and KH 2 PO 4 (≥99.5 %) were supplied by Sigma-Aldrich, 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was acquired from Serva. LDH activity assay was purchased from Promega (G7891). Human IL-1β ELISA Set II was obtained from BD Biosciences (557953). Penicillin/streptomycin, Dulbecco's modified Eagle's medium (DMEM), minimum essential medium (MEM), fetal bovine serum (FBS) and Dulbecco's phosphate-buffered saline (DPBS), were supplied by Biowest. Solvents THF, CH 2 Cl 2 and CH 3 CN were acquired from Scharlab, S.L. Distilled water was used to prepare the aqueous solutions. Hexanuclear cluster (Bu 4 N) 2 [-Mo 6 I 8 (CH 3 CO 2 ) 6 ] (1) was synthesized according to the procedure reported in the literature [72] (link).
9
Cytokine Quantification by ELISA
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IL-1β and IL-6 in the collected supernatants were measured with Human IL-1β ELISA Set II (BD Biosciences) and Human IL-6 Duoset ® ELISA (R&D Systems) as indicated in the manufacturer's protocols, respectively.
10
Cytokine Quantification and NFκB Activation
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Human IL-1β, IL-10 and TNFα concentrations in cell culture supernatants were determined employing the Human IL-1β ELISA Set II (BD Bioscience), the Human IL-10 ELISA Set (BD Bioscience) and the Human TNFα HTRF-Kit (Cisbio), respectively, according to the manufacturer's protocol. Stimulation with agonists was performed in RPMI-1640 + 10 % FBS 37 °C (8 h, 500 µl/well: IL-1β, IL-10; 5 h, 60 µl/well: TNFα). Reactions were stopped by aspiration of supernatants, which were subsequently frozen and stored (IL-1β, IL-10: -20 °C, TNFα: -80 °C) for later analysis. 2.5.7 NFκB luciferase assay THP-1-Lucia NFκB cells were seeded in 96-well plates and differentiated with PMA for 48 h. Stimulation with agonists was performed in M1-polarization medium (using RPMI-1640 without phenol red) for 48 h at 37 °C and 5 % CO2. To measure luciferase activity corresponding to NFκB activation, 20 µl/well of cell culture supernatant were transferred to a black 96-well plate, 50 µl/well of QUANTI-Luc assay solution (InvivoGen,) were added in the dark and luminescence was immediately measured using the EnVision multimode plate reader (Perkin Elmer).
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