Serum and wound effluent proteins were quantified using a Luminex 100/200 IS xMAP Bead Array Platform (Millipore Corp, Billerica, MA) as previously described (Hawksworth et al., 2009 (link)). Thirty-two cytokines, chemokines and growth factors were quantified using a Human Cytokine 30-plex panel supplemented with a custom Human 2-plex panel (Life Technologies; Cat. No LHC6003 and LCP0002, Grand Island, NY). Data were categorized as related to the initial débridement, the penultimate débridement, and the final débridement.
Human cytokine 30 plex panel
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Human cytokine 30-plex panel is a multiplex assay designed to simultaneously measure the concentrations of 30 different human cytokines and chemokines in a single sample. The panel allows for the quantitative analysis of multiple analytes from a small sample volume.
Automatically generated - may contain errors
18 protocols using human cytokine 30 plex panel
1
Serum and Wound Effluent Protein Quantification
2
Cytokine Profiling of MSC-PBMC Coculture
3
Co-culture of Calu-3 Cells and PBMCs
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For the co-culture studies, calu-3 cells were seeded and infected with 0.05 MOI of infectious virus and mock-infected cell control material in duplicate wells. The inoculum was removed 2 hours later and 500 µl of calu-3 media was added to each well. The media was removed and fresh media was added one day post infection (p.i.). One day later, i.e. 2 days p.i., trans-well inserts (polyester trans-well inserts, 0.33 cm with 0.4 u pores, Corning, Inc., Corning, NY) with one million live PBMCs (viability determined by trypan blue exclusion) were placed in the virus infected and control wells. Media was collected from the basolateral chamber at 6, 24, and 48 hours after addition of the PBMCs. The collected media was centrifuged, stored at −80°C, and later tested by multiplex luminex assays according to the manufacturer's instructions for FGF-Basic, IFN-γ, IL–12 (p40 / p70), IL–13, RANTES, MIP–1α, MIG, MIP–1β, VEGF, IL–1β, IL–2, IL–4, IL–5, IL–6, IL–2R, MCP–1, Eotaxin, IL–8, IL–10, IL–15, IL–17, IL–1RA, GM–CSF, G–CSF, EGF, HGF, TNF-α, IL–7, IP-10, IFN-α (Human cytokine 30-plex panel, Life technologies) and ENA-78, MCP-2 and IL-28A (Human cytokine 3 plex panel, Millipore).
4
Multiplex Cytokine Profiling of Supernatants
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Multiplex Cytokine Profiling of Cryopreserved Samples
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Cryopreserved (−80°C) samples were analyzed using Human cytokine 30-plex panel (Life Technologies, Carlsbad, CA) and FlexMAP 3D instrument (Luminex, Austin, TX). Data analysis was done using xPONENT software (Luminex).
6
Multiplex Cytokine Profiling of Cell Supernatants
7
Secretome Analysis of Organoid Cultures
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The used medium from the organoid culture was analyzed for secretome diversity and levels. Supernatants of conditioned organoid media were collected 3 days after a fresh medium change, followed by a brief centrifugation (500g for 5 minutes) and then stored at –80°C. Subsequently, medium supernatants were analyzed by magnetic bead-based multiplex Luminex assays for cytokines, chemokines, and growth factors, including fibroblast growth factor-basic, interferon-γ, IL12 (p40/p70), IL13, CCL5 (regulated on activation, normal T-cell expressed and secreted or CCL5 [RANTES]), CCL3 (MIP-1alpha), CXCL9 (monokine induced by gamma interferon or CXCL9 [MIG]), CCL4 (MIP-1beta), VEGF, IL1b, IL2, IL4, IL5, IL6, IL2R, CCL2 (MCP-1), CCL11 (Eotaxin), IL8, IL10, IL15, IL17, IL1RA, granulocyte-macrophage colony-stimulating factor, granulocyte-colony stimulating factor, epidermal growth factor, HGF, tumor necrosis factor alpha, IL7, CXCL10 (IP-10), and interferon-α (Human Cytokine 30-plex Panel; Life Technologies, Carlsbad, CA), according to the manufacturer’s instructions using Luminex xMAP (multi-analyte profiling) technology. Results were plotted as picograms per milliliter.
8
Multiplex Cytokine Analysis in Serum
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We used multiplex cytokine kits (Invitrogen Human Cytokine 30-Plex Panel, LHC6003) in order to measure serum levels of IL-1β, IL-5, IL-6, IL-7, IL-12, and TNF-α. Dose measurements were performed with the use of a Luminex 200 system (Luminex Corporation, Austin, TX, USA) in accordance with the manufacturer's specifications (Invitrogen Corporation, Carlsbad, CA, USA). The sensitivity of the test was specified by the manufacturer (Invitrogen Corporation, Carlsbad, CA, USA) in the informative material included in the kits.
The average sensitivity of the test for IL-1β was 5 pg/ ml, with an inter-assay variation coefficient of 4.8%. For IL-5, the average sensitivity of the test was 0.5 pg/ml with an inter-assay variation coefficient of 7.5%. The average sensitivity of the test for IL-6 was 0.5 pg/ml with an inter-assay variation coefficient of 7%. In the case of IL-7, the average sensitivity of the test was 10 pg/ml with an inter-assay variation coefficient of 9.8%. The sensitivity of the test for IL-12 was 1 pg/ml, and the inter-assay variation coefficient of 8.1%. The test for TNF-α revealed an average sensitivity of 0.5 pg/ml, with inter-assay variation coefficient of 8.3%.
The average sensitivity of the test for IL-1β was 5 pg/ ml, with an inter-assay variation coefficient of 4.8%. For IL-5, the average sensitivity of the test was 0.5 pg/ml with an inter-assay variation coefficient of 7.5%. The average sensitivity of the test for IL-6 was 0.5 pg/ml with an inter-assay variation coefficient of 7%. In the case of IL-7, the average sensitivity of the test was 10 pg/ml with an inter-assay variation coefficient of 9.8%. The sensitivity of the test for IL-12 was 1 pg/ml, and the inter-assay variation coefficient of 8.1%. The test for TNF-α revealed an average sensitivity of 0.5 pg/ml, with inter-assay variation coefficient of 8.3%.
9
Multiplex Plasma Cytokine Profiling
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Peripheral blood samples were taken by venous puncture and collected in sterile EDTA Vacutainers, between 8:00 and 10:00 a.m., they were processed immediately or within 2 hours after extraction. Plasma samples were obtained after high speed centrifugation for 10 minutes at 3,500–4,000 rpm and immediately aliquoted and frozen at -80°C for its conservation.
Plasma samples were clarified by high-speed centrifugation and analyzed using Luminex xMAP technology platform. The current investigation required the assembly of an extensive multiplex array consisting of cytokines, chemokines, soluble receptors, growth and angiogenic factors, which were evaluated using bead-based immunoassays. The selected array was the Human cytokine 30-Plex panel (Invitrogen) with the following analytes: IL-1β, IL-1RA, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-15, IL-17, TNF-α, IFN-α, IFN-γ, GM-CSF, MIP-1α, MIP-1β/CCL4, IP-10, MIG, Eotaxin/CCL11, Rantes/CCL5, MCP-1/CCL2, VEGF, G-CSF, EGF, FGF-basic, and HGF.
Plasma samples were clarified by high-speed centrifugation and analyzed using Luminex xMAP technology platform. The current investigation required the assembly of an extensive multiplex array consisting of cytokines, chemokines, soluble receptors, growth and angiogenic factors, which were evaluated using bead-based immunoassays. The selected array was the Human cytokine 30-Plex panel (Invitrogen) with the following analytes: IL-1β, IL-1RA, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-15, IL-17, TNF-α, IFN-α, IFN-γ, GM-CSF, MIP-1α, MIP-1β/CCL4, IP-10, MIG, Eotaxin/CCL11, Rantes/CCL5, MCP-1/CCL2, VEGF, G-CSF, EGF, FGF-basic, and HGF.
10
Multiplex Cytokine Profiling of Serum
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We used multiplex cytokine kits (Invitrogen Human Cytokine 30-Plex Panel, LHC6003) in order to measure serum levels of IL-4. Measurements were performed with a Luminex 200 system (Luminex Corporation, Austin, TX, USA) in accordance with the manufacturer's specifications (Invitrogen Corporation, Carlsbad, CA, USA). The sensitivity of the test was specified by the manufacturer (Invitrogen Corporation, Carlsbad, CA, USA). The average sensitivity of the test was < 0.5 pg/ml, with an inter-assay variation coefficient of 8.7%.
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