Collection of colon tissues and processing was performed as previously described (Henderson et al., 2015 (link)). Briefly, a 1 cm2 colon tissue biopsy was collected from ASF and CONV mice (n=4 per group), placed in PBS to remove intestinal contents and transferred to a tube containing RPMI plus gentamicin (50 μg ml−1), penicillin (200 IU ml−1) and streptomycin (200 μg ml−1) at room temperature with shaking at 280 rpm. After 30 min, tissues were transferred to 96-well plates containing RPMI plus gentamicin (50 μg ml−1), penicillin (200 IU ml−1), streptomycin (200 μg ml−1), L-glutamine (2 mM), 2-mercaptoethanol (0.05 mM) and pyruvate (1 mM), and incubated at 37°C in 5% CO2. After 24 h, supernatants were harvested and frozen at −20°C until further use. Levels of proinflammatory markers IL-1β, IL-12, IFNγ and TNFα were assessed using a multiplex bead assay and Bio-Plex 200 multiplex reader (Bio-Rad, Hercules, CA).
Bio plex 200 multiplex reader
Manufactured by Bio-Rad
Sourced in Belgium
The Bio-Plex 200 multiplex reader is a laboratory instrument designed for the detection and quantification of multiple analytes in a single sample. The core function of the Bio-Plex 200 is to perform multiplex assays, which allow the simultaneous measurement of various biomolecules, such as proteins, cytokines, or DNA, in a single experiment.
Automatically generated - may contain errors
Lab products found in correlation
Bio plex manager software v 6, by Bio-Rad (1 mentions)
Streptavidin phycoerythrin, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Anti il 22 pe, by Thermo Fisher Scientific (1 mentions)
Anti cd11b pe cy7, by Thermo Fisher Scientific (1 mentions)
Bio plex manager 6, by Bio-Rad (1 mentions)
Bio plex pro human chemokine panel 40 plex kit, by Bio-Rad (1 mentions)
Prism 6, by GraphPad (1 mentions)
5 protocols using bio plex 200 multiplex reader
1
Colon Tissue Biopsy and Cytokine Analysis
2
Multiplex Pneumococcal Antibody Assay
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Human antipneumococcal reference serum 007sp28 (link) and NHP study sera were precleared using pneumococcal cell wall polysaccharide adsorbents and then assayed using a custom multiplex bead array similar to those described elsewhere.29 (link),30 Briefly, samples were incubated with fluorescent magnetic microspheres (Luminex Corp) labeled with pneumococcal polysaccharides (FXImmune Diagnostics). After sample binding, microspheres were washed in PBS, 1% BSA (Invitrogen) and incubated for 30 minutes at room temperature with 4 μg/mL biotinylated antihuman/rhesus IgG Fc clone H2 (Southern Biotech). Microspheres were washed and labeled with 8 μg/mL streptavidin-phycoerythrin (Invitrogen). Beads were read on a BioPlex 200 multiplex reader (Bio-Rad) and analyzed using BioPlex Manager software v6.2 (Bio-Rad). NHP test sera IgG-binding of each polysaccharide was then calculated relative to that of 007sp reference serum.
3
Neutrophil IL-22 Production in Leishmaniasis
4
COVID-19 Cytokine and Chemokine Profiling
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The concentrations of 16 pro-inflammatory cytokines and chemokines were measured in plasma collected at D0 from 6 controls, 10 non-severe and 11 severe COVID-19 patients by multiparameter-based immunoassays (Milliplex Human Cytokine/chemokine/Growth factor panel A magnetic bead panel kit-Merck, Belgium) according to the manufacturer’s instructions. The samples were selected chronologically from patients with a clear status. The panel included granulocyte macrophage colony-stimulation factor (GM-CSF), interferon-alpha (IFN-α), interferon-gamma (IFN-γ), interleukin (IL)-1β, IL-6, IL-7, IL-8, IL-10, IL-17A, IL-22, interferon gamma-induced protein 10 (IP-10), monocyte chemo-attractant protein-1 (MCP-1), monokine induced by gamma interferon (MIG), macrophage inflammatory protein (MIP)-1α, MIP-1β, and tumor necrosis factor alpha (TNF-α). Results were analyzed with a Bio-Plex 200® Multiplex reader, Bio-Plex ManagerTM Manager 4.1 Software (BIO-RAD laboratories, Nazareth Eke, Belgium).
5
Multiplex Analysis of T84 Cell Cytokines
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Cell-free basolateral T84 supernatants were analyzed in duplicate using the Bio-Plex Pro™ Human Chemokine Panel, 40-Plex Kit (#171AK99MR2; Bio-Rad, Hercules, CA), and the Bio-Plex 200 Multiplex Reader (Bio-Rad), according to the manufacturer’s instructions. Concentration in range values were used for analysis to identify differences in cytokine expression among the uninfected and infected T84 cells. Samples from three independent experiments were analyzed in duplicate and graphed using GraphPad Prism 6 (GraphPad Software, La Jolla, CA). For samples where every value was out of range, indicating very low levels of cytokine production, a value of 0.1 was substituted for statistical analysis. The data are expressed as the means of the concentrations (pg/ml) ± standard errors of the mean (SEM). Statistical significance was assessed at a p value of <0.05 by one-way ANOVA with Tukey’s multiple comparisons test using GraphPad Prism 6.
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