Plasma samples were used to measure systemic levels of aspartate aminotransferase (AST) (#7036) and alanine aminotransferase (ALT) (#7018) (as markers of hepatocellular injury), creatinine (#7075) and urea (#7144) (as markers of renal dysfunction) using colorimetric clinical assay kits (FAR Diagnostics, Verona, Italy) according to the manufacturer’s instructions. Systemic cytokine levels were determined in plasma using the Luminex suspension bead-based multiplexed Bio-Plex Pro™ Mouse Cytokine Th17 Panel A 6-Plex (#M6000007NY) assay (Bio-Rad, Kabelsketal, Germany). The cytokines (IL-1β, IL-6, TNF-α, IFN-γ, IL-17 and IL-10) were measured following the manufacturer’s instructions.
Bio plex pro mouse cytokine th17 panel a 6 plex
Manufactured by Bio-Rad
Sourced in Germany
The Bio-Plex Pro™ Mouse Cytokine Th17 Panel A 6-Plex is a multiplex assay kit designed to quantify six mouse cytokines associated with the Th17 immune response. The kit utilizes magnetic bead-based technology to enable the simultaneous detection and measurement of multiple analytes in a single sample.
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6 protocols using bio plex pro mouse cytokine th17 panel a 6 plex
1
Plasma Biomarkers of Organ Injury
2
Cytokine Production in Wt and Was-/- Mice
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Cytokine production was evaluated on serum samples from wt and Was−/− mice before and after administration of LPS, CpG, apoptotic cells, or infection with LCMV, by using the Bio-Plex Pro Mouse Cytokine Th17 Panel A 6-plex (IL6, IL17a, IFNg, IL1b, IL10, TNFa) (Biorad) and following the manufacturer’s instruction. The plate was acquired using the Bio-Plex MAGPIX Multiplex Reader (Biorad).
3
Quantification of Plasma Cytokine Levels
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Cytokine assays (Bio-Plex Pro™ Mouse Cytokine Th17 Panel A 6-Plex; Bio-Rad Laboratories, Hercules, CA) were performed according to manufacturer's instructions to quantify levels of interleukin (IL)-6, IL-1β, tumor necrosis factor (TNF)-α, interferon (IFN)-γ, IL-17A, and IL-10 in plasma. Briefly, samples were diluted with sample diluent at 1:4. Samples and supplied standards were transferred to a 96-well plate containing antibodies coupled to magnetic beads for each analyte and incubated on a shaker for 30 min at room temperature (RT). Samples were washed three times with supplied wash buffer and then incubated with detection antibodies on a shaker for 30 min at RT. Samples were washed three times with supplied wash buffer and then incubated with Streptavidin-PE on a shaker for 10 min at RT. Samples were washed three times with supplied wash buffer. Beads were resuspended for 5 min and then quantified on a Bio-Plex® 200 system (Bio-Rad Laboratories). All samples were run in duplicate, and duplicate values were averaged.
4
Multiplex Biomarker Profiling in Plasma
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Cytokines were determined in plasma by using the Luminex suspension bead-based multiplexed Bio-Plex Pro™ Mouse Cytokine Th17 Panel A 6-Plex (#M6000007NY) assay (Bio-Rad, Kabelsketal, Germany). The cytokines IFN-γ, IL-1β, IL-6, IL-10, IL-17 and TNF-α were measured according to the manufacturer’s instructions. Plasma hormone levels of PAI-1 (#ab197752) and Resistin (#ab205574) were measured through conventional enzyme-linked immunosorbent assays (ELISA) according to the manufacturer’s instructions.
5
Multiplex Cytokine Profiling of Myofibroblast-ENS Cocultures
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Multiplex ELISA was used to determine soluble cytokine concentrations from myofibroblasts and ENS in both apical and basolateral chambers of cocultures. Media was collected after 48 hours of incubation on day 3 before further functional analyses or fixation. A Bio-Plex Pro™ Mouse Cytokine Th17 Panel A 6-Plex (Bio-Rad Laboratories, Inc) was used to quantify production of IFN-γ, IL-1β, IL-6, IL-10, IL-17A, and TNF-α. Singleplex ELISAs were performed for MIP-2 and TGF-β1 (ProcartaPlex, Invitrogen). TGF-β1 was activated immediately before analysis via a 10 minute incubation with 1N Hydrochloric Acid (Fisher Sci) and subsequent neutralization with 1.2N Sodium Hydroxide/0.5M HEPES. All cytokine assays were analyzed on a Luminex 200 system (Bio-Rad Laboratories, Inc) according to manufacturer instructions.
6
Cytokine Profiling in BALF Using Bio-Plex
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A Bio-Plex Pro Mouse Cytokine Th17 Panel A 6-Plex (Bio-Rad, Hercules, CA, USA) was used to evaluate the cytokine levels in BALF supernatant. Interferon (IFN)-γ, interleukin (IL)-1β, IL-6, IL-10, IL-17A and tumor necrosis factor (TNF)-α were measured according to the manufacture's protocol. Concentration of each cytokine was determined and calculated using a beads assay on the Bio-Rad Bio-Plex 200 System (Bio-Rad) and Bio-Plex Manager version 6.0 software, respectively.
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