Purified human B cells were cultured in RPMI 1640 with 5% FBS, 2mM L-glutamine, 50μM 2-betamercaptoethanol, 10mM HEPES, and 50μg/ml gentamicin at a concentration of 2.9–3.1 × 106 cells/ml. B cells were stimulated with: 1) CpG oligodeoxynucleotides (ODN) 2395 (a TLR9 agonist; Hycult Biotech, Plymouth Meeting, PA) and 1μg/ml plus BCR stimulation using rabbit anti-human IgM Ab fragment (Jackson ImmunoResearch, West Baltimore Pike, PA) at a concentration of 2μg/ml; 2) PAM3CSK4 (a TLR1/2 agonist; Invivogen, San Diego, CA) at a concentration of 10μg/ml; 3) CpG-ODN targeting TLR9 at a concentration of 10μg/ml. B cells were plated in round bottom inert grade 96-well plates (Thermo Fisher Scientific, Waltham, MA) and cultured in duplicate for 2 days and the supernatant was collected. Supernatant IL-6, IL-10, and TNFα concentrations were measured using Luminex Assay kits (Thermo Fisher Scientific) following the manufacturer’s instructions. IgM and IgG levels were assayed with ELISAs (Abcam, USA) in response to BCR+TLR9 stimulation on day 3 post-activation. B cell proliferation relied on a CyQUANT cell proliferation assay kit (Eugene, OR) per the manufacturer’s instructions.
Luminex assay kits
Manufactured by Thermo Fisher Scientific
Sourced in United States
Luminex assay kits are a multiplex detection platform that utilizes color-coded magnetic microspheres to measure multiple analytes simultaneously in a single sample. The core function of these kits is to enable the quantitative analysis of a variety of biomolecules, including proteins, peptides, and nucleic acids.
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Lab products found in correlation
4 protocols using luminex assay kits
1
Profiling Cytokine and Antibody Responses of Activated Human B Cells
2
Multiplex Cytokine and Antibody Profiling
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Serum levels of multiple cytokines and antibody subtypes were measured using the Luminex assay kits (Thermo Fisher, USA). Measurements were performed according to the manufacturer’s instructions and analysed on the Luminex platform.
3
Multiplex Cytokine and Antibody Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Serum levels of multiple cytokines and antibody subtypes were measured using the Luminex assay kits (Thermo Fisher, USA). Measurements were performed according to the manufacturer’s instructions and analysed on the Luminex platform.
4
Luminex Antibody Multiplex Assay
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Serum levels of multiple antibody subtypes were measured using the Luminex assay kits (Thermo Fisher Scientific, Inc.; cat. no. EPX070-20815-901). Measurements were performed according to the manufacturer's instructions. Diluted serum samples were added onto 96-well plates coated with magnetic beads and incubated for 120 min after vortexing. The beads were then washed, and the detection antibody mixture was added and incubated for 30 min at room temperature. After incubation and plate washing, the samples were analyzed on the Luminex™ platform (Thermo Fisher Scientific, Inc.).
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