BAL cell pellets were re-suspended in cell lysis buffer (BioRad, Hercules, CA), sonicated for 20 seconds, and passed through a 1.2 μm filter before total protein concentration was determined by colorimetric assay (BioRad). The levels of 27 immunomodulating proteins were then assayed in BAL cell lysates using Bio-Rad Multiplex bead array following manufacturer guidelines as previously described (Bird et al., 2010 (link)). All samples were assayed in duplicate and the results were analyzed using the Bio-Plex manager software, version 5.0. All values were normalized to total protein concentration and reported as picograms of protein of interest per milligram of total protein.
Multiplex bead array
Manufactured by Bio-Rad
The Multiplex bead array is a laboratory instrument designed for the simultaneous detection and quantification of multiple analytes or biomarkers in a single sample. It utilizes color-coded beads coated with specific capture agents to enable multiplexed assays, providing efficient and high-throughput analysis.
Automatically generated - may contain errors
6 protocols using multiplex bead array
1
Multiplex Immunomodulatory Protein Assay
2
Cytokine Quantification in Salmonella Cultures
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IFN-γ and IL-10 concentrations in the supernatants of Salmonella WCL-stimulated cultures were measured by ELISA according to the manufacturer's protocol (BD Biosciences, CA). Similarly IL17A concentration was measured by ELISA according to the manufacturer's protocol (eBiosciences). For some experiments supernatants collected from the culture assays were assayed for Th1 cytokines by use of a multiplex bead array (Bio-Rad, CA) and were analyzed using the Bioplex workstation and associated software (Bio-Plex Manager, version 6.1; Bio-Rad).
3
Activated T Cell Cytokine Profiling
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T cells were incubated at a concentration of 4 × 106 cells/ml in a 48-well plate which was pre-coated with αCD3 antibody (145-2C11). Supernatants were harvested after 48 h in culture. Cytokine production was measured using a Millipore Multiplex bead array following manufacturer's instructions and analyzed by Luminex (Bio-Rad) reader.
4
ELISA-Based Cytokine Profiling
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PGE2 levels in BALF and cell culture supernatants were measured by ELISA (Cayman Chemical). NHBE cells were stimulated with TGFβ (5 ng/ml), TNFα (10 ng/ml), EGF (50 ng/ml), IL-1β (1 ng/ml), or LPS (100 ng/ml) for 6 h at 37 °C. Cytokines in BALF supernatants were analyzed using a customized MultiPlex bead array (Bio-Rad) as previously described (7 (link)).
5
Lung T Cell Stimulation Assay
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Lungs from mice were isolated, cut with scissors into small pieces, and digested with 2 mg/mL Collagenase IV at 37-degrees Celsius for 45 min. Tissue was strained through nylon filters to isolate single cell suspension. T cells from lungs of mice were restimulated by co-culture irradiated (24 gray) B6 splenocytes that were loaded with antigen (1 μg/mL SEA) and were incubated at 37°C in complete medium DMEM, 10% fetal bovine serum (HyClone Characterized, Thermo Fisher, Cat# SH30396, Lot# KTH31760), 1% β-mercaptoethanol, and antibiotics. After 48 hrs, plates were directly frozen without separating supernatant from stimulated cells. Total cytokine production was measured using a Millipore Multiplex bead array following manufacturer's instructions and analyzed by a Luminex (Bio-Rad) reader.
6
Cytokine Profiling in Murine Plasma
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