Protein expression analysis was performed using two methods as depicted in Figure 1 . The first was a BD BioSciences cytometric bead array Th1/Th2/Th17 kit that served as an exploratory step to determine the concentration of interleukin (IL-2), IL-4, IL-6, IL-10, TNF, IFN-γ, and IL-17A cytokines. The assay was done on 31 AP patients (15 MAP, 11 MSAP, and 5 SAP) and 6 healthy control donor samples on days 1, 3, 5, and 7 post epigastric pain (see the supplementary section for detailed protocol). The second analysis was done using a MILLIPLEX® MAP Human Th17 Magnetic Bead Panel kit (Millipore™, Massachusetts, United States).
Milliplex map human th17 magnetic bead panel kit
Manufactured by Merck Group
Sourced in United States
The MILLIPLEX® MAP Human Th17 Magnetic Bead Panel kit is a multiplex assay designed for the quantitative measurement of multiple analytes related to T helper 17 cells. The kit utilizes magnetic beads coated with capture antibodies specific to the target analytes, allowing for the simultaneous detection of multiple biomarkers in a single sample.
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6 protocols using milliplex map human th17 magnetic bead panel kit
1
Cytokine Expression Analysis in Acute Pancreatitis
2
Plasma Cytokine Profiling in HBV-ACLF
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Plasma levels of sST2 were determined by enzyme-linked immunosorbent assay (ELISA) using commercially available kits (R&D Systems; Minneapolis, MN, USA). Plasma samples from the 48 HBV-ACLF patients, 12 CHB controls, and 16 healthy controls were diluted 100-fold, 40-fold, and 20-fold, respectively, according to the manufacturer's instruction and results of preliminary experiments. Plasma levels of IL-33, TNF-α, IFN-γ, and IL-10 were determined simultaneously using the Luminex xMAP multiplex system with the Milliplex MAP human Th17 magnetic bead panel kit (Millipore Corporation, Billerica, MA, USA) [30 (link), 31 (link)]. The assays were performed on a Bio-Plex 200 system (Bio-Rad, Hercules, CA, USA). Each sample was measured in duplicate. Detection limits were 5 pg/mL (IL-33), 3 pg/mL (TNF-α), 10 pg/mL (IFN-γ), and 1 pg/mL (IL-10).
3
Characterizing Th17 Immune Responses
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The different aspects of the study included different numbers of patients as illustrated (Figure 1 ). From the 40 patients, plasma and cell samples were processed in the laboratory within 4 h of phlebotomy. Plasma from a total of 31 out of 40 patients was analysed using the Th1/Th2/Th17 cytometric bead array (CBA) kit in an initial exploratory study. Based on this analysis, plasma samples from 23 patients were randomly selected for analysis of selected Th17 related cytokines including IL-6 using the MILLIPLEX® MAP Human Th17 Magnetic Bead Panel kit (Millipore™, Massachusetts, United States).
RNA was extracted from peripheral blood mononuclear cells (PBMCs) using the TriReagent® (Sigma Aldrich, Missouri, United States) method from 13 patients for screening of genes with the human innate and adaptive RT2 Profiler 96-well PCR array plates (QIAGEN, Hilden, Germany). Findings showed dose-dependent expression of the CCR8 gene with disease severity, prompting further analysis in 29 patients using quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) to verify its roles. To characterize cell types into monocytes, lymphocytes, and granulocytes, seven patients were included in an antibody specific multicolour immunophenotyping flow cytometry experiment.
RNA was extracted from peripheral blood mononuclear cells (PBMCs) using the TriReagent® (Sigma Aldrich, Missouri, United States) method from 13 patients for screening of genes with the human innate and adaptive RT2 Profiler 96-well PCR array plates (QIAGEN, Hilden, Germany). Findings showed dose-dependent expression of the CCR8 gene with disease severity, prompting further analysis in 29 patients using quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) to verify its roles. To characterize cell types into monocytes, lymphocytes, and granulocytes, seven patients were included in an antibody specific multicolour immunophenotyping flow cytometry experiment.
4
Cytokine Profiling in Type 1 Diabetes
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Cytokine levels in EDTA-treated plasma from controls and T1D patients were measured by the xMAP Technology on Luminex 200 (Luminex Corp., Austin, TX). Levels of 20 different cytokines were determined with the Milliplex MAP High Sensitivity Human Cytokine kit (n = 20 controls, n = 34 T1D patients) and the Milliplex MAP Human Th17 Magnetic Bead Panel kit (n = 20 controls, n = 36 T1D patients; Millipore Corp., Billerica, MA) (Table 2 ). Cytokine levels were analyzed in accordance with the manufacturer’s protocol, with levels below the detection limit being imputed as 10% less than the minimum detectable concentration limit, as calculated by the manufacturer’s protocol.
5
Plasma Th17 Cytokine Profiling Protocol
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A fasting blood sample (5 mL) was drawn from each case (within 48 h of admission and before any treatment) and control (at the time of interview) and was centrifuged at 2000 g/minute for 15 minutes at 4 °C within 2 hours. Then, the plasma was aliquoted into 500-μL straws and frozen at −80 °C immediately after processing until use. Plasma levels of Th17-related cytokines, including IL-17A, IL-17F, IL-21, IL-22 and IL-6, along with IFN-γ, IL-10, IL-9 and IL-4 were measured by MILLIPLEX MAP Human Th17 Magnetic Bead Panel kits (Millipore, Billerica, MA., USA.) based on the Luminex xMAP technology (Luminex Corporation, Austin, TX., USA.). Plates were run on the Luminex MagPix machine (Luminex Corporation) and data were collected using Luminex xPONENT 4.2 software and were analysed using MILLIPLEX Analyst 5.1 software (Millipore). Concentrations of cytokines (pg/ml) were calculated using a standard curve. All samples were measured once. Two replicate quality control samples were run with each assay (replicate QC1 samples, low level; replicate QC2 samples, high level). The coefficients of variation (CVs) of replicate quality control samples were <10% for all cytokines.
6
Serum IL-17A and IL-17E Quantification
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The serum samples were collected from pSS patients or healthy controls with polypropylene microfuge tubes. These samples were stored at -80°C without repeated thawing and freezing, and were not thawed until measurement. IL-17A and IL-17E in these sera were determined with MILLIPLEX MAP Human Th17 magnetic bead panel kits (Millipore, USA), following instructions from the manufacturer. Data were read on a Luminex-200 machine (Luminex Corporation, USA) and analyzed with MILLIPLEX Analyst 5.1 software (Millipore, USA).
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