Mouse sera were collected with SST Tubes (BD Microtainer), centrifuged for 10 min, then stored at −80°C. The concentration of human cytokines was determined after 2-fold serum dilution, by using a custom Magnetic Luminex Performance Assay (R&D Systems, USA). Procedures were performed according to the manufacturer's instructions. Results were acquired on Bio-Plex System and analyzed with Bio-Plex Manager Software 4.0 (Biorad Laboratories).
Magnetic luminex performance assay
Manufactured by R&D Systems
Sourced in United States
The Magnetic Luminex Performance Assay is a multiplex immunoassay system that uses magnetic microspheres coated with capture antibodies to detect and quantify multiple analytes simultaneously in a single sample. The system utilizes Luminex technology, which combines flow cytometry and laser detection to provide accurate and sensitive measurements of analyte concentrations.
Automatically generated - may contain errors
Lab products found in correlation
Bio plex manager software 4, by Bio-Rad (2 mentions)
Bio plex 200 system, by Bio-Rad (2 mentions)
Bio plex data pro, by Bio-Rad (2 mentions)
Bio plex system, by Bio-Rad (1 mentions)
Pierce bicinchoninic acid bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Luminex xmap technology, by Merck Group (1 mentions)
Milliplex map human sepsis magnetic bead panel 3, by Merck Group (1 mentions)
Platelia sars cov 2 total antibody assay, by Bio-Rad (1 mentions)
Human luminex discovery assay, by R&D Systems (1 mentions)
Sample activation kit 1, by R&D Systems (1 mentions)
C3a human procartaplex simplex kit, by Thermo Fisher Scientific (1 mentions)
Bio plex manager software v 6, by Bio-Rad (1 mentions)
Bio plex 200, by Bio-Rad (1 mentions)
Bio plex 200tm system, by Bio-Rad (1 mentions)
Magnetic luminex assay, by R&D Systems (1 mentions)
Quantikine elisa, by R&D Systems (1 mentions)
20 protocols using magnetic luminex performance assay
1
Quantification of Mouse Cytokines
2
Multiplex Analysis of Inflammation Cytokines
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Inflammation cytokines in the cell-culture supernatants were assessed by a Magnetic Luminex Performance Assay (R&D Systems) following the manufacturer’s guidelines. A Luminex X-200 (Luminex, Austin, TX) was used to read the multiplex assay. Absolute cytokine concentrations were determined using 5-parameter logistic curve fits in the R&D Analyte Software.
3
Quantifying Plasma Cytokine Profiles in NPC
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Plasma from the patients with NPC was isolated within 2 h after blood collection and frozen at −80 °C for later use. The Magnetic Luminex Performance assay (R&D) was applied to evaluate TNF‐a, VEGF, GM‐CSF, IL‐12p70, IL‐10, IL‐8, IL‐6, IL‐4, IL‐2, and IL‐1β. We used the ELISA Kit purchased from the R&D company to determine plasma TGF‐ β1 level. All detection assays were performed according to the manufacturer's protocols.
4
Quantifying Metalloproteinases in Tissue Samples
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The concentrations of MMP-1, MMP-2, MMP-3, MMP-9 and MMP-13 in tissue homogenates
were assessed using a Magnetic Luminex Performance Assay (R&D Systems,
Inc.) 5-Plex Panel. Multiplexes were run on a LABScanTM 100 platform (Luminex
Corp., Austin, TX, USA) equipped with Luminex® 100 IS software.
Tissue homogenates were incubated with antibody-coated microspheres, which bind to
specific MMPs. Microsphere–MMP complexes were washed and incubated with
biotinylated MMP antibodies, which bind to MMPs present on the microspheres. A
final incubation was performed in which phycoerythrin-labeled streptavidin was
allowed to bind to biotinylated MMP antibodies present on microspheres.
Microspheres were then loaded into a LABScanTM 100 analyzer, which quantifies the
amount of phycoerythrin fluorescence present on each of the distinct microsphere
groups. At least 50 individual microspheres were counted for each MMP, and the
median fluorescence intensity was used for subsequent calculations.
were assessed using a Magnetic Luminex Performance Assay (R&D Systems,
Inc.) 5-Plex Panel. Multiplexes were run on a LABScanTM 100 platform (Luminex
Corp., Austin, TX, USA) equipped with Luminex® 100 IS software.
Tissue homogenates were incubated with antibody-coated microspheres, which bind to
specific MMPs. Microsphere–MMP complexes were washed and incubated with
biotinylated MMP antibodies, which bind to MMPs present on the microspheres. A
final incubation was performed in which phycoerythrin-labeled streptavidin was
allowed to bind to biotinylated MMP antibodies present on microspheres.
Microspheres were then loaded into a LABScanTM 100 analyzer, which quantifies the
amount of phycoerythrin fluorescence present on each of the distinct microsphere
groups. At least 50 individual microspheres were counted for each MMP, and the
median fluorescence intensity was used for subsequent calculations.
5
Biomarkers of Immune and Endothelial Activation
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Biomarkers of immune and endothelial activation were evaluated in EDTA anticoagulated plasma stored at − 80 °C using a custom Magnetic Luminex® Performance Assay (R&D Systems) [19 (link), 24 (link)]. The markers assessed included Cystatin C, C-X-C motif chemokine Ligand 10 (CXCL10) / interferon γ-induced protein 10 kDa (IP-10), chitinase-3-like protein 1 (CHI3L1), soluble tumor necrosis factor receptor-1 (sTNFR1), soluble triggering receptor expressed on myelocytes (sTREM-1), interleukin 6 (IL-6), and interleukin 8 (IL-8) (representing immune activation), as well as angiopoietin-2 (Angpt-2), angiopoietin-1 (Angpt-1), soluble fms-like tyrosine kinase-1 (sFlt-1), soluble vascular cell adhesion molecules (VCAM-1), and soluble intercellular adhesion molecule-1 (sICAM-1) (representing endothelial activation).
6
Inflammatory Cytokine Profiling in BAL Fluid
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Inflammatory cytokine (IL-6, IL-8, TNF-α, IL-1β, macrophage chemoattractant protein-1, IL-10, IL-1ra, and vascular endothelial growth factor) content of pooled patient BAL fluid was measured by a commercially available custom Magnetic Luminex® Performance Assay (R&D Systems, UK) as per the instructions of the manufacturer. Protein concentration in pooled patient BAL fluid was measured using the Pierce™ BCA (Bicinchoninic Acid) Protein Assay Kit (ThermoFisher Scientific, Waltham, MA, USA) as per the instructions of the manufacturer.
7
Serum Biomarker Profiling in Sepsis
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The concentration of serum molecules was determined by using Luminex technology. MILLIPLEX MAP Human Sepsis Magnetic Bead Panel 1 (macrophage migration inhibitory factor [MIF], soluble intercellular adhesion molecule 1 [sICAM-1], soluble Fas [sFas], soluble Fas ligand [sFasL], soluble vascular cell adhesion molecule 1 [sVCAM-1], and total plasminogen activator inhibitor-I [tPAI-I]) (Millipore, Billerica, MA, USA), MILLIPLEX MAP Human Sepsis Magnetic Bead Panel 3 (lactoferrin, elastase 2, neutrophil gelatinase–associated lipocalin [NGAL], resistin, and thrombospondin-1) (Millipore, Billerica, MA, USA) and Magnetic Luminex Performance Assay (Human High Sensitivity Cytokine Base Kit A, interferon-γ [IFN-γ]) (R&D Systems, Minneapolis, MN, USA) were used for determining the serum levels of these molecules according to the manufacturer’s instructions. Data was acquired on Luminex xMAP technology (Millipore, St Charles, MO, US). For concentration calculation, the calibration curve for each serum molecule was analyzed with a five parameter logistic curve fit curve through the Milliplex Analyst Software (Viagene Tech, Carlisle, MA, USA).
8
Quantitative Analysis of MMPs in γ-FFs and γ-CAFs
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Quantitative determination of MMP-1, MMP-9, and MMP-13 released by γ-FFs and γ-CAFs were performed by Magnetic Luminex® Performance assay (R&D Systems). γ-FFs and γ-CAFs were plated for 4 h in D-MEM supplemented with 10% FCS and D-MEM/F-12:1/1 supplemented with 10% FCS, respectively at 37°C in a 5% CO2 incubator. γ-FFs were incubated for 2 h at 37°C in presence of 100 ng/mL TNFα in D-MEM supplemented with 1% FCS without antibiotics while γ-CAFs were directly incubated in D-MEM/F-12:1/1 supplemented with 1% FCS without antibiotics. Both γ-FFs and γ-CAFs were maintained for 5 d in their respective medium supplemented with 1% FCS (without any medium change). Supernatants (duplicate) were analyzed with a Magnetic Luminex® Performance assay multiplex kit including human MMP-1, MMP-9, and MMP-13. Data were acquired on Bio-Plex™ 200 System (Bio-Rad Laboratories) and analyzed on Bio-Plex Manager (version 6.1).
9
Biomarkers of Immune and Endothelial Activation
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Two panels of biomarkers were designed to interrogate host pathways of: (1) immune activation; and (2) endothelial activation. Selected molecules have been previously validated as biologically plausible and clinically informative markers host response to infection. Immune activation was assessed using circulating (plasma) levels of C-X-C motif chemokine Ligand 10 (CXCL10)/interferon γ-induced protein 10 kDa (IP-10) [25 (link),26 (link)], chitinase-3-like protein 1 (CHI3L1) [42 (link)], soluble tumor necrosis factor receptor-1 (sTNFR1) [41 (link)], soluble triggering receptor expressed on myelocytes (sTREM-1) [40 (link)], interleukin 6 (IL-6), and interleukin 8 (IL-8) [41 (link)]. Endothelial activation was assessed using angiopoietin-2 (Angpt-2) [34 (link),35 (link)], angiopoietin-1 (Angpt1) [27 (link)], soluble fms-like tyrosine kinase-1 (sFlt-1) [30 (link)], soluble vascular cell adhesion molecules (VCAM-1) [30 (link)], and soluble intercellular adhesion molecule-1 (sICAM-1) [30 (link)]. Biomarkers of immune and endothelial activation were evaluated using a custom Magnetic Luminex® Performance Assay (R&D Systems) in EDTA anticoagulated plasma stored at −80 °C until testing [51 (link)].
10
Inflammatory Markers in COVID-19 Serology
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CRP and fibrinogen levels were analyzed by the hospital laboratory. Cytokine assays were performed in the cancer institute research laboratory. Serum levels of IL-6, IL-8, IL-10, TNFα, GM-CSF, and IFNγ were measured using a Magnetic Luminex Performance Assay (R&D Systems) according to the manufacturer's protocol [22 ]. Data were analyzed using Bio-Plex Manager Software and GraphPad Prism. COVID-19 serology was assayed at baseline, day 7 and day 14 utilizing Bio-Rad's Platelia SARS-CoV-2 total antibody assay.
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