The blood samples were collected from the tail vein of rOmpB-4-CMR group, rOmpB-4 group, or CMR group mice and pooled together to obtain a serum sample on days 7, 14, or 21 after primary immunization. And IFN-γ and TNF-α in the serum sample were determined using a Luminex Bio-Plex 200 IS 100 instrument (BIO-RAD, Hercules, CA) with multiplex kits and related reagents produced by Affymetrix (Santa Clara, CA).
Multiplex kits
Manufactured by Thermo Fisher Scientific
Sourced in United States
Multiplex kits are laboratory instruments designed to analyze multiple analytes or targets simultaneously in a single sample. These kits enable efficient and high-throughput screening of various biological markers or molecules of interest.
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Lab products found in correlation
6 protocols using multiplex kits
1
Cytokine Profiling in Immunized Mice
2
Quantifying Antibody and Cytokine Responses
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Three mice from each immunization group were sacrificed on day 14 after treatment, and serum were separated from whole blood. Antibodies against C. burnetii WCA, recombinant proteins, or peptides were measured by ELISA as described previously [24] (link). Briefly, 100 µl of antigen solution (2 µg/ml) was applied to each well to coat the microplates (Corning, Corning, NY), and each serum sample was diluted 1∶50. The optical density (OD) of each well was read at 450 nm using a UVM340 microplate reader. A multiplex immunoassay in a Luminex Bio-Plex 200 IS 100 instrument (BIO-RAD, Hercules, CA) was also used to quantify serum cytokines including interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-10 (IL-10), interleukin-12p70 (IL-12p70), IFN-γ, and TNF-α. All measurements were performed in at least triplicate, as described previously [24] (link), [25] (link). Multiplex kits and related reagents were purchased from Affymetrix (Santa Clara, CA).
3
Multiplex Cytokine and MMP Profiling in CSF
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Multiplex assays were run by the Bursky Center for Human Immunology & Immunotherapy Programs (CHiiPs) at Washington University School of Medicine according to the manufacturer’s instructions. Frozen supernatant CSF was slowly thawed and then analyzed in duplicate with multiplex kits (Thermofisher Scientific, Waltham, MA) for the following pro-inflammatory cytokines: IL-1β, IL-2, IL-5, IL-6, IL-8, IL-12, IL-17, TNF-α, GM-CSF, IFN-γ; and anti-inflammatory cytokines: IL-4 and IL-10; as well as selected MMPs: MMP-2, MMP-3, MMP-9, MMP-7 (catalog number EPXP130-10100-901, EPX03A-10829-901, PPX-01-MMP7). Briefly, magnetic beads were added across all the wells on the plate; CSF samples and standards were then added in duplicate. Following washing steps, the detection antibody was added followed by streptavidin incubation. Beads were then resuspended with reading buffer and data were acquired on a Luminex system. The concentration of each antigen was calculated by plotting the expected concentration of the standards against the multiplex fluorescent immunoassay generated by each standard. A 4-parameter logistic regression was used for the best fit curve. Protein concentration is reported as pg/mL for each analyte.
4
Multiplex Cytokine Quantification
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The concentrations of IL-1 , IL-6, IL-10, IL-17A, IL-17F, IL-22, IL-23, IL-33, IFN- , and TNF- were measured using commercial Multiplex kits (ThermoFisher) according to the instructions of the manufacturer.
5
Quantifying Immune Response Cytokines
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Culture supernatants collected 24 h and one week post stimulation were assayed using Multiplex kits (Thermo Fisher Scientific, USA). The specific kit used for 24 h culture supernatant contained the following analytes: IL-10, IL-6, IL-8, IL-29, CCL-2, TNF-α, IFN-α, CXCL-10, CCL-19, IL-18, TREM-1, IL-1Ra, GM-CSF, IL-15, and PDGF-BB. The kit for seven-day supernatants contained the analytes IL-29, TNF-α, IFN-α, IL-6, IFN-γ, IL-17 A, IL-1β, and granzyme B. The procedure was performed according to the manufacturer’s protocol. Briefly, the supernatant was mixed with premixed beads overnight, and after incubation with detection antibodies and streptavidin-PE for 1 h each, the plate was run on Magpix to identify specific cytokines.
6
Cytokine Profiling of Cell Cultures
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Culture supernatants collected 24 h and 1 week post stimulation were assayed using Multiplex kits (Thermo Fisher Scientific, USA). The specific kit used for the 24-h culture supernatant contained the following analytes: IL-10, IL-6, IL-8, IL-29, CCL-2, TNF-α, IFN-α, CXCL-10, and CCL-19. The kit for the 7-day supernatants contained these analytes: TNF-α, IFN-α, IL-6, IFN-γ, IL-17A, IL-1β, IL-22, and granzyme B. The procedure followed was according to the protocol of the manufacturer. Briefly, the supernatant was mixed with premixed beads (30 cytokines) overnight, and after incubation with detection antibodies and streptavidin-PE for 1 h each, the plate was run on Magpix to identify specific cytokines. IL-18 was detected using specific ELISA kit (Boster Bio, CA, USA).
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