Supernatants collected at the relevant time points and stored at –80°C were filter-sterilized no more than 24 h prior to analysis by multiplex bead-based immunoassay. Immune Monitoring 65-Plex Human ProcartaPlex Panel (Invitrogen) was used according to the manufacturer’s recommendations and acquired on a Luminex Bio-Plex 200 platform and Bio-Plex Manager 6.0 software (Bio-Rad). Culture medium was measured in parallel to the samples to assess any potential residual presence of the target biomolecules in the human serum. Results were analyzed using R package nCal.
Luminex bio plex 200 platform
Manufactured by Bio-Rad
The Luminex Bio-Plex 200 platform is a multiplex assay system that uses flow cytometry technology to simultaneously detect and quantify multiple analytes in a single sample. The platform utilizes color-coded magnetic beads coated with specific capture antibodies to enable the measurement of various proteins, cytokines, and other biomolecules in a high-throughput manner.
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Lab products found in correlation
4 protocols using luminex bio plex 200 platform
1
Multiplex Cytokine Profiling
2
Multiplex Cytokine Profiling in Macrophages and Plasma
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A ProcartaPlex Mix&Match bead array (ThermoFischer scientific) was used to quantify granulocyte macrophage colony-stimulating factor (GM-CSF), IL-1β, IL-10, IL-12p70, IL-1RA, IL-23, IL-6, IL-8 (CXCL8), IP-10 (CXCL10), monokine induced interferon gamma (MIG/CXCL9), monocyte inflammatory protein 1 alpha and beta (MIP-1α/β), tumor necrosis factor alpha (TNF-α) and HGMB-1 within 0.2 μm-filtered macrophages’ culture supernatants. An independent ProcartaPlex Mix&Match bead array was used to assess the presence of GM-CSF, interferon gamma (IFN-γ), IL-1α, IL-1β, IL-10, IL-12p70, IL-17F, IL-1RA, IL-23, IL-4, IL-6, IP-10, macrophage chemoattractant protein 1 (MCP-1/CXC L), MIP-1β, and TNF-α in plasma samples from patients that were frozen at – 80 °C after collection, thawed and then centrifuged at 1000 × g for 5 min to remove aggregates before processing. Both assays were performed following manufacturer’s instructions. Data were acquired on a Luminex Bio-Plex 200 platform and Bio-Plex Manager 6.0 software (Bio-Rad). Cytokine and chemokine concentrations were interpolated from standard curves using the nCal R package.
3
Quantifying Secreted Senescence-Associated Factors
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Conditioned media (CM) was collected by culturing cells or islets in growth media lacking FBS for 18–24 h. CM was collected, clarified by centrifugation (3000 g for 5 min at room temperature) and either used immediately or stored at 4 °C for up to 2–3 weeks. Luminex assays were carried out on CM from cells treated as indicated using a custom magnetic bead kit that included mouse SASP factors IL-6, Serpine1 and Igfbp3, or human SASP factors CXCL1, CXCL8/IL-8, IGFBP4, GDF-15, or TNFRSF10C (R&D systems). 50 μl of CM was assayed on a Bio-Plex 200 luminex platform (Bio-Rad) as previously [7 (link),14 (link)] and secreted amounts in 1–2 ml of CM were normalized to viable cell counts to calculate amount secreted per 100,000 cells or for human islets secretion was normalized to total RNA content. Alternatively, media was spin-concentrated using a 3 kDa MWCO centrifugal unit (Millipore-Sigma) and the concentrated conditioned media was analyzed by Luminex.
4
Multiplex Cytokine Profiling of Cryopreserved Plasma
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Plasma was stored at −80°C for immunology assays. Using the Bio-Plex Pro Human Cytokine Standard 27-Plex kit (Group I) on the Biorad Bioplex 200 Luminex platform the following analytes were measured: interleukin (IL)-1β, IL-1 receptor antagonist (IL-1Ra), IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12p70, IL-13, IL-15 IL-17A, eotaxin, granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF/CSF2), interferon gamma (IFN-γ), monocyte chemoattractant protein-1 (MCP-1)/C-C motif chemokine ligand 2 (CCL2), macrophage inflammatory protein-1 alpha (MIP-1α/CCL3), MIP-1 beta (MIP-1β/CCL4), platelet-derived growth factor-BB (PDGF), regulated on activation, normal T cell expressed and secreted (RANTES/CCL5), tumor necrosis factor-alpha (TNF-α), and vascular endothelial growth factor (VEGF). Biomarkers were chosen based on commercially available pre-mixed kits and included parameters previously described to be involved in TB pathogenesis.34 (link),67 (link)
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