After 24-h incubation with samples in 96-well plates, the following cytokines production from each well containing 10,000 cells was analyzed: interleukin (IL)-6; IL-10, tumor necrosis factor (TNF)-α; granulocyte colony-stimulating factor (G-CSF); granulocyte macrophage colony-stimulating factor (GM-CSF); lipopolysaccharide-induced CXC chemokine (LIX; CXCL5); monocyte chemotactic activating factor (MCP)-1; macrophage inflammatory protein (MIP)-1α, MIP-1β, MIP-2, RANTES (CCL5; regulated on activation, normal T cell expressed and secreted), vascular endothelial growth factor (VEGF); interferon gamma-induced protein 10 (IP-10; CXCL10); and leukemia inhibitory factor (LIF). Cytokines were measured using a Luminex assay based on xMAP technology with multiplex cytokine assay kits and a Bio-Plex 200 suspension array system (Bio-Rad, Hercules, CA, USA), as described previously [4 (link),5 (link),6 (link),7 (link),8 (link),9 (link)]. The assay used in this experiment was designed for the multiplexed quantitative measurement of multiple cytokines in a single well, using as little as 50 µL of cell culture supernatant. Baicalein, a well-known anti-inflammatory flavonoid, was used as a positive control.
Multiplex cytokine assay kit
Manufactured by Bio-Rad
Sourced in United States
The Multiplex cytokine assay kit is a laboratory equipment designed for the simultaneous detection and quantification of multiple cytokines in a single sample. It utilizes bead-based multiplex technology to allow for the efficient analysis of a wide range of cytokines in a cost-effective and high-throughput manner.
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7 protocols using multiplex cytokine assay kit
1
Multiplexed Cytokine Profiling of Cell Cultures
2
Multiplex Cytokine Profiling Assay
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This assay was performed with multiplex cytokine assay kits and Bio-Plex 200 suspension
array system (Bio-Rad) as described previously (Lee et al., 2011). The following cytokine
productions were analyzed: interleukin (ILHo, IL-φ, IL-6, IL-10, granulocyte
macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor
(G-CSF), macrophage colony-stimulating factor (M-CSF), macrophage inflammatory protein
(MIP)-^ MIP-^, MIP-2, CXCL10 (IP-10), monocyte chemotactic activating factor
(MCP)-1, RANTES, leukemia inhibitory factor (LIF; IL-6 class cytokine),
lipopolysaccharide-induced CXC chemokine (LIX; CXCL5), and tumor necrosis factor -α
(TNF-α).
array system (Bio-Rad) as described previously (Lee et al., 2011). The following cytokine
productions were analyzed: interleukin (ILHo, IL-φ, IL-6, IL-10, granulocyte
macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor
(G-CSF), macrophage colony-stimulating factor (M-CSF), macrophage inflammatory protein
(MIP)-^ MIP-^, MIP-2, CXCL10 (IP-10), monocyte chemotactic activating factor
(MCP)-1, RANTES, leukemia inhibitory factor (LIF; IL-6 class cytokine),
lipopolysaccharide-induced CXC chemokine (LIX; CXCL5), and tumor necrosis factor -α
(TNF-α).
3
Serum Cytokine Profiling in Animal Study
4
Cytokine Profiling in Plasma Samples
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The protein levels of various pro-inflammatory cytokines/chemokines; RANTES, IL-6, IL-8, monocyte chemotactic protein-1 (MCP-1), IL1-β, and TNF-α were measured using multiplex cytokine assay kit (Bio-Rad, CA, USA) as per the manufacturer’s protocol. Briefly, plasma samples were centrifuged at 3000 g for 10 min and the supernatants were diluted with three volumes of sample diluent. Next, 50 μl of each sample and standards were mixed with magnetic beads and incubated on a shaker at room temperature for 30 min. The beads were washed and 25 μl of detection antibody was added to each well followed by incubation for 30 min at room temperature. Furthermore, the samples were washed and incubated with 50 μl streptavidin-PE conjugate for 10 min. Finally, 125 μl of the assay buffer was added and the samples were analyzed using Biorad Bioplex HTS (Bio-Rad, CA, USA). The concentrations of the cytokines were determined with Bio-plex manager 5 using 5-PL statistics using standard curve.
5
Plasma Cytokine Profiling by Multiplex Assay
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Plasma levels of various pro-inflammatory cytokines/chemokines including RANTES, IL-6, IL-8, MCP-1, and TNF-α were measured as described previously [19 (link)] using the multiplex cytokine assay kit (Bio-Rad, CA, USA). Briefly, plasma samples were centrifuged and the supernatants were diluted with sample diluent (1:3). Next, 50 µl of each sample was used to determine the levels of these cytokines/chemokines using Biorad Bioplex HTS (Bio-Rad, CA, USA). The levels of cytokines were determined using the standard curve.
6
Profiling Cytokine Secretion in Co-cultured Immune Cells
7
Multiplex Cytokine Profiling
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