AML cells (1 × 106 cells/mL in 1 mL/well) were seeded in 24-well plates with StemSpan SFEMTM medium supplemented with the cytokines G-CSF, SCF, and FLT3-L and treated with one of the five SYK inhibitors. StemSpan SFEMTM medium supplemented with cytokines G-CSF, SCF, and FLT3-L was treatment-free control. Supernatants were harvested after 48 h and stored at −80 °C until analyzed. Human Magnetic Luminex Assays (R&D Systems; Minneapolis MN, USA) were used to analyze all supernatant samples according to the manufacturer’s protocol. Cytokine release was estimated as a ratio to untreated control for each cytokine and SYK inhibitor.
Human magnetic luminex assay
Manufactured by R&D Systems
Sourced in United States, France, United Kingdom
The Human Magnetic Luminex Assay is a multiplex immunoassay platform designed for the simultaneous quantification of multiple analytes in a single sample. The assay utilizes magnetic beads coated with capture antibodies specific to the target analytes. After incubation with the sample, the beads are washed, and a biotinylated detection antibody is added. The signal is then generated through the addition of streptavidin-conjugated fluorescent proteins.
Automatically generated - may contain errors
Lab products found in correlation
Bio plex 200, by Bio-Rad (6 mentions)
Bio plex 200 system, by Bio-Rad (5 mentions)
Bio plex manager 5, by Bio-Rad (5 mentions)
Magpix, by Merck Group (4 mentions)
Bio plex magpix multiplex reader, by Bio-Rad (3 mentions)
Human th1 th2 cytokine procartaplex panel, by Thermo Fisher Scientific (2 mentions)
Bio plex manager software version 6, by Bio-Rad (2 mentions)
Quantikine elisa human igf 1 immunoassay, by R&D Systems (2 mentions)
Bio plex manager software, by Bio-Rad (2 mentions)
Duoset elisa kit, by R&D Systems (2 mentions)
Cobas ampliprep cobas taqman, by Roche (1 mentions)
Bio plex 200 assay reader, by Bio-Rad (1 mentions)
Milliplex analyst software v 3, by Merck Group (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
U plex kit, by Mesoscale (1 mentions)
Luminex 200, by Thermo Fisher Scientific (1 mentions)
Meso quickplex sq 120, by Mesoscale (1 mentions)
Chi3l1, by R&D Systems (1 mentions)
Thrombospondin 1, by Abcam (1 mentions)
Cxcl5, by Abcam (1 mentions)
69 protocols using human magnetic luminex assay
1
Evaluating SYK Inhibitor Cytokine Effects
2
Multiplex Protein Biomarker Profiling in Plasma
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Whole blood was collected in heparin tubes, centrifuged at 2500 rpm for 10 min for plasma separation and stored at −80 °C. Plasma samples tested in different batches were diluted 1:2, and the levels of plasma biomarkers were measured using commercial kits, Human Magnetic Luminex assays (Lot number L129636 R&D, Minneapolis, MN, USA), as previously published [29 (link)]. The kit detected 15 soluble proteins, namely, IFN-gamma, NCAM, TNF-alpha, IL-8, IL-10, IL1b, GM-CSF, IL-13, IL-12, CD40-ligand, MMP-1, MMP-2, MMP-8, S100A8 and MPO. The sample plates were read on the same day using a MAGPIX system, xMAP instrument (Luminex, Austin, TX, USA). Finally, xPONENT software, version 4.3 (Luminex, Austin, TX, USA), was used for bead acquisition and analysis.
3
Protein Quantification in Human ALF
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Protein levels in human ALF were determined using custom Human Magnetic Luminex Assays (R&D Systems), Human TH1/TH2 Cytokine ProcartaPlex Panel (ThermoFisher Scientific), and by Human SP-A, SP-D and C3 ELISA (LifeSpan Biosciences, Inc., Seattle, WA) per kit instructions.
4
Cytokine and Adipokine Profiling in Fasting Blood Samples
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Blood samples were drawn from the participants in the morning after ≥ 8 h of fasting and stored at -80 °C. C-reactive protein (CRP) was analyzed by standard techniques at the Sahlgrenska University Hospital. Serum levels of cytokines and adipokines were assessed at BL and M6. The serum levels of the cytokines (TNF-α, IL-1β, IL-6, IL-8, IL-12/IL-23 p40, IL-13, IL-17, IL-23, interferon (IFN)-γ, and the adipokines (leptin (analyzed at twofold dilutions), resistin, total (tot)-adiponectin (200-fold dilution) and High Molecular Weight (HMW)-adiponectin (50-fold dilution)) were measured with Human Magnetic Luminex® Assays (R&D-systems) at the Department of Rheumatology and Inflammation research, Gothenburg University, according to the manufacturer's instructions. The analysis and quantification were performed using a Bio-Plex 200 system (Bio-Rad) with five-parameter logistic standard curves. Samples with analyte levels below the detection level were excluded from the analysis.
5
Cytokine and IgA Detection Assays
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Cytokines in HLH, HLAC and AIM assay supernatants were measured by Human Magnetic Luminex Assays using a Premixed Multiplex assay (for detection of IFN-γ, IL-2, IL-4, IL-6, IL-17A, IL-21, IL-22, TNF-α) from R&D Systems according to the manufacturer's recommendations. Samples were diluted 1:2. Acquisition was performed on a MAGPIX system and data were analyzed with Milliplex Analyst or xPONENT software (Luminex, Merck Millipore). For sample sets in which IL-6 concentrations exceeded the standard, IL-6 was measured by IL-6 ELISA (Thermofisher Scientific) in accordingly diluted samples as per manufacturer's instructions. Secreted IgA was measured by ELISA (IgA human uncoated ELISA kit, Thermofisher Scientific) in appropriately diluted samples as per manufacturer's instructions.
6
Protein Quantification in Human ALF
7
Biomarkers for Hepatitis B Assessment
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The biochemical and hematological parameters were routinely detected by standard methods in local hospitals. Serum HBV DNA (range 2.0 × 101–1.7 × 108 IU/ml) was measured by the COBAS AmpliPrep/COBAS TaqMan (Roche Diagnostics, Basel, Switzerland). Serum HBsAg (range of 20–52,000 IU/ml) was quantified using the Roche Elecsys HBsAg II assay (Roche Diagnostics, Penzberg, Germany). The serum levels of YKL-40 were determined using Human Magnetic Luminex® Assays (LXSAHM-08, R&D Systems, Inc, Minneapolis, MN, USA) according to the manufacturer’s instructions. The serum concentrations of hyaluronic acid (range of 2–200 μg/L), laminin (5–900 μg/L), were measured using a chemiluminescence immunoassay kit (Yuande Bio-Medical Engineering Co., Ltd, Beijing, China).
8
Blood Biomarkers in 12-Month Follow-up
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Venous blood samples were collected in a fasting state between 07:00 and 08:00 AM following the baseline sleep study and at a 12-month follow-up visit. Plasma samples were aliquoted and stored at −70 °C.
Levels of Ang-2, sRAGE, Tie-2, VEGF-A, and E-Selectin were measured in 30 μL of plasma diluted 1:2 using Human Magnetic Luminex® Assays (R&D Systems, catalog# LXSAHM) according to the manufacturer's instructions. The data were collected on a BD FACSAria III flow cytometer and analyzed by Flowjo. The mean fluorescence intensity of each pro-inflammatory biomarkers of the standards was used for calculating the standard curve for each biomarker using a log–log curve fit. Average Coefficient of Variance for Protein Standard across all five analytes (intra-plate measurements) was 6.0% and for Replicated Sample across all five analytes (inter-plate measurements) 11.3%.
There are no missing data for the primary analyses of the levels of biomarkers as only participants with available blood sample were included in this secondary analysis.
Levels of Ang-2, sRAGE, Tie-2, VEGF-A, and E-Selectin were measured in 30 μL of plasma diluted 1:2 using Human Magnetic Luminex® Assays (R&D Systems, catalog# LXSAHM) according to the manufacturer's instructions. The data were collected on a BD FACSAria III flow cytometer and analyzed by Flowjo. The mean fluorescence intensity of each pro-inflammatory biomarkers of the standards was used for calculating the standard curve for each biomarker using a log–log curve fit. Average Coefficient of Variance for Protein Standard across all five analytes (intra-plate measurements) was 6.0% and for Replicated Sample across all five analytes (inter-plate measurements) 11.3%.
There are no missing data for the primary analyses of the levels of biomarkers as only participants with available blood sample were included in this secondary analysis.
9
Magnetic Luminex Assay for Secreted Proteins
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Proteins of interest were determined in supernatants using custom Human Magnetic Luminex® Assays (R&D biosystems) for analysis of COL1α1, MMP-1, MMP-2, TIMP-1, VEGFA, VEGFC, platelet-derived growth factor (PDGF) DD, IL-6Rα and IL-1R1 according to manufacturer’s instructions. Standard curves were generated from provided analyte standards in the ranges outlined in Supplementary Table S3 . SM was used as a blank. Sample dilutions were determined from previous experiments and are listed in Supplementary Table S3 . Assays were measured on the Bio-Plex 200 assay reader and concentrations were calculated using the Bio-Plex Manager Software, version 6.2 (both Bio-Rad, Hercules, CA, USA).
10
Blood Biomarker Profiling Protocol
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As part of the examinations, a fasting blood sample (no anti-coagulant) was collected from each participant. Following clotting, the samples were centrifuged, and the serum supernatants were collected within 1 hour after collection. The resulting serum samples were stored at -80°C until analysis. Blood biochemical parameters were measured using an auto-analyzer (JCS-BM1650, Nihon Denshi, Tokyo, Japan). Human Magnetic Luminex Assays (R&D Systems, Minneapolis, MN, USA) were used to measure serum proteins. Sample dilution and reagent adjustments were performed according to the kitspecific protocol; a MAGPIX xPONENT 4.2 (Luminex, Austin, TX, USA) was used as the measuring instrument.
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