Opteia mouse il 6 elisa set

Macrophages were seeded in 96-well tissue culture plates (45 × 10³/well) in 100 µl culture macrophage differentiation medium. The following day, macrophages were stimulated in triplicates with the reagents of interest for 36 h. Finally, either cell viability was determined using the MTT assay or upregulation of IL-6 and Il-1β production was measured by enzyme-linked immunosorbent assay (ELISA) of supernatants (R&D: mouse IL-1β/IL-1F2 DuoSet, #DY401; BD Bioscience: OptEIA mouse IL-6 ELISA Set, #555240). In each cell viability experiment, one triplicate of cells was treated with a cytotoxic cocktail containing 1 µg/ml Fc-CD95L, 20 µM CHX, and 1% (w/v) sodium azide, which triggers complete cell death. The triplicate of cells challenged with the cytotoxic mixture defined 0 % viability and the triplicate of untreated cells defined 100% viability. All other viability measurements were normalized against these values. The averages of the technical triplicates were considered as a data point for further statistical evaluation (one-way analysis of variance Bonferroni’s multiple comparison test function; GraphPad Prism5 software from GraphPad software, La Jolla, CA, USA).

IL-6 was quantified in lung homogenate using the OptEIA mouse IL-6 ELISA set (BD) following the manufacturer’s directions. Briefly, homogenates were diluted 1:25 in assay diluent prior to analysis. Homogenates from three mice were assayed in triplicate for each strain infection. Statistical significance was determined by one-way ANOVA, followed by Bonferroni’s posthoc test (***, P ≤ 0.001).

RAW264.7 cells were cultured in 24-well plate with 500 μl medium. Cells were then treated with 10 μg/ml MVs for 30 h. Culture supernatant was removed and centrifuged at 2,000 g for 10 min. The supernatant was diluted with 10% FBS/PBS. Cytokine concentration was quantified using ELISA kits, OptEIA Mouse IL-6 ELISA Set, OptEIA Mouse TNF ELISA Set (both from BD Biosciences), and Mouse IL-1 beta SimpleStep ELISA Kit (Abcam), according to manufacturer’s protocol.

Three days poststimulation with Spike_RBD 15-mer peptides, cell supernatants were collected and used for ELISA to measure IFNγ, IL-6, IL-2, and TNFα production. IFNγ, IL-2, IL-6, and TNFα were measured using the BD OptEIA Mouse IFN-γ ELISA Set (BD Biosciences, 555138), BD OptEIA Mouse IL-6 ELISA Set (BD Biosciences, 555240), BD OptEIA Mouse IL-2 ELISA Set (BD Biosciences, 555148), and Mouse TNFα Uncoated ELISA kit (Invitrogen, 88-7324-22) per manufacturer's protocol.

ELISAs and sequential ELISA measurements were performed as described previously^{5 (link),6 (link),27 (link)}. The following ELISA kits were used according to the manufacturer’s recommendations: BD OptEIA Mouse IL-6 ELISA Set and BD OptEIA Mouse MCP-1 ELISA Set (both BD Pharmingen, San Diego, CA, USA), Mouse JAM-A DuoSet ELISA and Mouse Complement Component C5a DuoSet ELISA (both R&D Systems, Minneapolis, MN, USA), Mouse Occludin ELISA Set (Wuxi Donglin Sci&Tech Development, Jiangsu, P.R.C.), and Mouse Mucin 2 / MUC2 (Sandwich ELISA) ELISA Kit (LSBio, Seattle, WA, USA). Activated complement component C3a was detected using an in-house sandwich ELISA employing antibodies purchased from BD Pharmingen and a protein standard purchased from R&D Systems. The measured concentrations of parameters in the jejunum and colon tissue homogenates were calculated relative to the total protein concentration in the corresponding sample.

The BD OptEIA™ Mouse IL-6 ELISA Set (BD Biosciences, San Diego, CA, USA; BD 555240) was used to measure the levels of inflammatory cytokines IL-6, IL-1β, TNFα, IFNγ and IL-17A in the mouse serum. Absorbance at 450 nm was measured on a BioTek^® Synergy HT Microplate reader (Santa Clara, CA, USA). The serum levels of IL-1β, TNFα, IFNγ, and IL-17A were measured at LABISKOMA (Seoul, Republic of Korea) via multiplex analysis.