Culture media of imDCs, mDCs, and OVA peptide-treated spleen cells were collected. The levels of cytokines and chemokines, including interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interleukin-12 (IL-12), interferon-γ (IFN-γ), interferon-β (IFN-β), and interleukin-10 (IL-10), were measured by flow cytometry using LEGENDplex™ bead-based immunoassay kits (BioLegend). The levels of TNF-α and IL-10 in the culture media of adenovirus-infected and LPS-treated TOT/Tmod1-/- imDCs and TLR agonist-treated imDCs were measured using Enzyme linked immunosorbent assay (ELISA) kits (BOSTER Biotechnology, Wuhan, China).
Legendplex bead based immunoassay kit
Manufactured by BioLegend
Sourced in United States
The LEGENDplex bead-based immunoassay kit is a multiplex assay that can simultaneously measure multiple analytes in a single sample. The kit uses color-coded beads coated with capture antibodies specific to each target analyte. After the sample is incubated with the beads, a detection antibody is added, and the resulting immune complexes are quantified using a flow cytometer.
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5 protocols using legendplex bead based immunoassay kit
1
Cytokine and Chemokine Profiling of Dendritic Cells
2
Dendritic Cell-Mediated CD4+ T Cell Activation
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Mature DCs were incubated with 10 μg/ml OVA peptide (323–339) (Alpha Diagnostic Intl Inc., San Antonio, TX, USA) at 37°C for 2 h. The cells were washed twice and resuspended in serum-free RPMI 1640 medium. Treated mDCs (1 × 106) were injected intravenously into C57BL/6J mice on days 1, 3, and 5. On day 8, the mice were sacrificed, and their spleen cells were collected, and re-stimulated with 10 μg/ml OVA peptide (323–339) at 37°C for 3 days (41 (link)). Then, the spleen cells were washed with PBS, stained with FITC-conjugated anti-mouse CD4 antibody, and further analyzed by flow cytometry. The fluorescence intensity indicated the proliferation of CD4+ T cells. The concentrations of IFN-γ and IL-10 in culture medium of OVA-peptide-treated spleen cells were measured by flow cytometry using LEGENDplex™ bead-based immunoassay kits (BioLegend).
3
Quantifying Cytokine Levels in Suppression Assay
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The levels of the cytokines IL-2 and IFN-γ in the culture supernatants from suppression assay were measured using LEGENDplex bead-based immunoassay KIT (Biolegend), according to the recommended procedure. Briefly, the samples were incubated with a panel of capture beads, then mixed with biotinylated detection antibody and subsequently with streptavidin–phycoerythrin, providing flourescent signals that were quantified on a flow cytometer. The concentrations of the cytokines were determined using a standard curve generated in the same assay. The experiments were performed in quadruplicate and repeated two times.
4
Multiplex Cytokine Profiling in Plasma
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Cytokines were measured in plasma samples, including IFN-β, IL-18 (ELISA, R&D Systems, Minneapolis, MN), IL-1β (Cytometric bead array flex kit, BD Biosciences, San Jose, CA), TNF, IL-6, and IFN-γ (LEGENDplex bead-based immunoassay kit, BioLegend, San Diego, CA).
5
Quantifying SARS-CoV-2 Antibody Levels
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Blood was drawn into vacutainer tubes containing citrate and plasma generated by centrifugation at 1000 x g for 10 min at 4 °C, followed by a second centrifugation of the supernatant at 10,000 x g for 10 min at 4 °C [15] . Plasma samples were aliquoted and stored at − 80 °C until further use.
Antibodies against the SARS-CoV-2 spike protein S1, nucleocapsid (NC)-, and the Receptor Binding Domain (RBD)-protein were determined by a LEGENDplex™ bead-based immunoassay kit (BioLegend, United States) according to the manufacturer´s instructions. Briefly, plasma was incubated with capture beads for 2 h while mixing. After washing, samples were incubated with biotinylated detection antibodies for 1 h forming capture bead-analyte-detection antibody complexes. Subsequently, streptavidin-phycoerythrin (SA-PE) was added for 30 min providing a fluorescent signal. Samples were measured on a Cytoflex S cytometer within 3 h and analyzed using LEGENDPlex v8.0 software (https://www.biolegend.com/en-us/legendplex). Absolute concentrations of anti-spike S1, anti-NC and anti-RBD IgGs (ng/ml) were assessed via a standard (BioLegend) concentration curve.
Antibodies against the SARS-CoV-2 spike protein S1, nucleocapsid (NC)-, and the Receptor Binding Domain (RBD)-protein were determined by a LEGENDplex™ bead-based immunoassay kit (BioLegend, United States) according to the manufacturer´s instructions. Briefly, plasma was incubated with capture beads for 2 h while mixing. After washing, samples were incubated with biotinylated detection antibodies for 1 h forming capture bead-analyte-detection antibody complexes. Subsequently, streptavidin-phycoerythrin (SA-PE) was added for 30 min providing a fluorescent signal. Samples were measured on a Cytoflex S cytometer within 3 h and analyzed using LEGENDPlex v8.0 software (https://www.biolegend.com/en-us/legendplex). Absolute concentrations of anti-spike S1, anti-NC and anti-RBD IgGs (ng/ml) were assessed via a standard (BioLegend) concentration curve.
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