For detection of murine autoantibodies by ELISA, microtiter plates were coated with 10 μg/mL nucleosomes isolated from apoptotic cells as described previously (73 (link)), 50 μg/mL cardiolipin (MilliporeSigma, C1649), and 10 U/mL Sm/RNP (GenWay Biotech, GWB-A1CECE) or precoated with 20 μg/mL poly-l -lysine (MilliporeSigma, P2658) before adding 20 μg/mL calf thymus DNA (MilliporeSigma, D3664). Sera were 1:50 or 1:25 diluted in PBS/2% FBS (Thermo Fisher Scientific, 26140079). Bound IgG was detected with HRP-conjugated goat anti-mouse IgG(H+L) (Southern Biotech, 1031-05) at 1:4,000 dilution, followed by substrate solution (Roche, 11112422001). Results were expressed as fold-changes relative to a designated positive sample. IFN-α level in peritoneal lavages and cell culture supernatants was quantified by cytometric bead array (BioLegend, 740633).
Cytometric bead array
Manufactured by BioLegend
Sourced in United States
The Cytometric Bead Array is a multiplex assay platform that enables the simultaneous detection and quantification of multiple analytes in a single sample. It utilizes fluorescently-labeled beads with capture antibodies specific to the target analytes. This allows for the measurement of various proteins, cytokines, or other biomolecules in a complex biological sample.
Automatically generated - may contain errors
Lab products found in correlation
Multi analyte flow assay kit, by BioLegend (2 mentions)
Hrp conjugated goat anti mouse igg h l, by Southern Biotech (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Safire2 plate reader, by Tecan (1 mentions)
Pierce bca protein assay, by Thermo Fisher Scientific (1 mentions)
Polytron homogenizer, by Kinematica (1 mentions)
Human il 1β elisa kit, by Cusabio (1 mentions)
Multiplex luminex assay, by BD (1 mentions)
7 protocols using cytometric bead array
1
Murine Autoantibody Detection by ELISA
2
Multiplex Cytokine Profiling Assay
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The concentrations of IL-36α, IL-36β, IL-36γ, IL-36Ra, and IL-1β were measured using a commercial human ELISA kit (CUSABIO, China). The IL-2, IL-4, IL-6, IL-9, IL-10, IL-13, IL-17 A, IL-17 F, IL-22, IFN-γ, and TNF-α concentrations were determined using the Multi-Analyte Flow Assay Kit (Biolegend, USA) with a Cytometric Bead Array (CBA). The above assay steps were performed according to the manufacturer’s recommended protocol.
3
Quantification of Mouse Serum S100A9
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Mouse serum was collected via cardiac puncture, clotted in room temperature for 30 min and collected by centrifuging twice at 10,000 g for 10 min at 4 °C. Lung proteins were extracted in RIPA lysis buffer with Protease Inhibitor Cocktails (Sigma) and homogenized with POLYTRON homogenizer (Kinematica) on ice. Protein levels were quantified by Pierce BCA protein assay (Thermo Scientific) as per the manufacturer’s instructions. All plates were read on a Safire II plate reader (Tecan).
Mouse S100A9 ELISA kit (R&D systems) was used to determine the level of S100A9/MRP14 in serum and lung lysate samples following manufacturer’s instructions. Cytokine concentrations were determined using Cytometric Bead Array (Biolegend) following the manufacturer’s manual.
Mouse S100A9 ELISA kit (R&D systems) was used to determine the level of S100A9/MRP14 in serum and lung lysate samples following manufacturer’s instructions. Cytokine concentrations were determined using Cytometric Bead Array (Biolegend) following the manufacturer’s manual.
4
Plasma Cytokine Profiling Protocol
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The plasma levels of IL-1β, IL-6, IL-8, IL-18, IL-23, TNF-α were detected by cytometric bead array, according to the manufacturer’s protocol (Biolegend). Data were obtained by flow cytometry and analyzed by FCS Express 6 Plus provided by the company. The value below the detection limit on each well was set to 0.
5
Quantitative Detection of Sputum Biomarkers
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Quantitative detection of sputum TIPE2 and sputum IL-1β was performed using the commercial human TNFAIP8L2 ELISA Kit (Catalog Number EK6248-2, Signalway Antibody, USA) and Human IL-1β ELISA Kit (CSB-E08053h, CUSABIO, Wuhan, China) according to the manufacturers’ protocols. The levels of other cytokines (IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-17A, IL-17F, IL-22, IL-23, TNF-α, and interferon (IFN)-γ) were measured using the Cytometric Bead Array under the Multi-Analyte Flow Assay Kit (Biolegend, USA) following the manufacturer’s instructions. All incubation steps were performed at room temperature and protected from light. LEGENDplex v8.0 software was used to analyze the data.
6
Cytokine Profiling of Organoid Supernatants
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Supernatants from PRR‐stimulated lung and intestinal organoids were used to determine the cytokine expression profiles using the LEGENDplex™ Human Inflammation Panel [13‐plex including IL‐1β, IFN‐α2, IFN‐γ, TNF‐α, MCP‐1 (CCL2), IL‐6, IL‐8 (CXCL8), IL‐10, IL‐12p70, IL‐17A, IL‐18, IL‐23 and IL‐33] cytometric bead array (BioLegend, San Diego, CA, USA), according to the manufacturer's protocol.
7
Silica-Induced Inflammatory Cytokine Profile
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Mice were exposed to 50 μl suspensions of 5000 μg of silica crystals in PBS by direct orotracheal instillation. Control mice received PBS. Animals were sacrificed 3 days after instillation and a bronchoalveolar lavage (BAL) was carried out by repeatedly instillating and withdrawing 1 ml of 1% BSA/PBS solution three consecutive times. Cytokine levels of BAL were measured using a Multiplex Luminex assay (BD sciences). Reagents for quantitative ProcartaPlex Luminex immunoassay were sourced from Affymetrix eBioscience. Cytometric Bead Array (Bio-legend) were used according to the manufacturer’s instructions. and results were read on the Bio-Plex 200 instrument. In order to set up the heatmap, the heatmap value of same cytokines was calculated as a fraction of the highest detected cytokines levels relative to the detected cytokines levels.
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