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51 protocols using human premixed multi analyte kit

1

Multiplex Biomarker Analysis of Whole Blood

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Peripheral venous whole blood samples were used to analyze the biomarkers of interest via magnetic Luminex multiplex bead array by using Luminex® 100/200™, Human Premixed Multi-Analyte kit (R&D Systems, Minneapolis, MN, USA) and Human Cardiovascular Disease Magnetic Bead Panel 3 MILLIPLEX® MAP kit (EMD Millipore Corporation, Billerica, MA, USA) according to the manufacturers’ instructions. The PCT and ENG were analyzed and quantified with the Human Premixed Multi-Analyte kit (R&D Systems), whereas the CRP and AGP were analyzed and quantified with the Human Cardiovascular Disease Magnetic Bead Panel 3 MILLIPLEX® MAP kit (EMD Millipore Corporation). The samples were evaluated using a MAGPIX® multiplexing system (Luminex Corporation, Austin, TX, USA), which is a fluorescent bead-based instrument. Results were analyzed and interpreted using the Luminex xPONENT® software (Luminex Corporation).
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2

Serum Biomarker Quantification Protocol

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Serum samples were collected and stored at − 80 °C freezer until analysis. Human Premixed Multi-Analyte Kit was purchased from R&D Systems (Minneapolis, Minnesota, USA). Serum CXCL13 and TGF-β were measured by Bio-Plex® suspension array system (Bio-Rad Laboratories Inc., California, USA). All samples were measured in duplicate. Four serum samples were excluded from this analysis as the volume were not enough for analysis. Two CXCL13 detection results were also excluded as they were reported with warning after bio-plex analysis. Eventually, 61 results of CXCL13 and 63 results of TGF-β were included in the following analysis.
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3

Multiplex Assay for Maternal-Fetal Chemokines

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Plasma levels of CXCL-4, CXCL-13, CXCL-16, and CCL-24 were measured by magnetic luminex screening assay, using Human Premixed Multi-Analyte Kit (R&D Systems, Inc. Minneapolis, MN, USA). Human, magnetic, premixed, microparticle cocktail of antibodies specific to CXCL-4, CXCL-13, CXCL-16, and CCL-24 was used to simultaneously screen maternal peripheral, placental and neonate cord plasma. The assay was carried out according to manufacturer’s instructions. Briefly, plates were incubated on the horizontal shaker with 50 μL/well of different diluted samples and concentration of standard chemokines provided alongside with 50 μL of human, magnetic, premixed, microparticle cocktail with antibodies specific for each chemokine. After washing using magnetic plate separator (Luminex, Austin, TX, USA, Cat# CN-0269-01), plates were incubated with 50 μL/well of human premixed biotin-antibodies cocktail specific for each chemokine. Antibody chemokine complexes were revealed using Streptavidin-PE. Plates were read using a Luminex MAGPix Analyzer (XMAP Technology, SN, USA) and results expressed as median fluorescence intensity (MFI). A standard curve was generated for each chemokine to convert MFI into corresponding chemokines relative concentrations.
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4

Magnetic Luminex Cytokine Quantification

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Cytokine quantification was performed as described8 (link). All analytes were measured by magnetic luminex screening assay using Human Premixed Multi-Analyte Kit (R&D Systems) according to the manufacturers’ instructions. Measurements were performed with a Bioplex200 Analyzer equipped with Bio-Plex ManagerTM Software (Bio-Rad).
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5

Cytokine Measurement in ILC2 Cultures

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After treatment for 4 hours, concentrations of selected cytokines in the supernatants of ILC2 (approximately 6 × 105 cells/well) cultures were measured with a Human Premixed Multi-Analyte Kit (R&D Systems, Minneapolis, Minn) with magnetic beads, according to the manufacturer's instruction. Results were obtained with a Bio-Plex 200 System (Bio-Rad Laboratories, Hercules, Calif).
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6

Multiplex ELISA for Frozen Supernatants

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Cell culture supernatant was frozen at −80°C until use for analysis. Multiplex ELISA was performed with human premixed multi-analyte kit (RandD Systems, USA) on a MAGPIX instrument (Luminex, USA). Data was analyzed with the xPONENT 4.2 software (Luminex, USA).
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7

Magnetic Luminex Assay Protocol

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Magnetic Luminex assays were performed according to the manufacturer's recommendations using a custom Human Premixed Multi‐Analyte Kit (R&D systems).
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8

Assessing Anti-SARS-CoV-2 Antibodies and Cytokines in Breastmilk

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The specimens were thawed and they were allowed to slowly reach room temperature. Then, they were vortexed for 20 s. For milk degreasing, we centrifuged the tubes at 5000× g for 25 min at 4 °C. The skimmed milk was transferred into a new tube for further processing.
All breastmilk samples were checked for the presence of reactive anti-S1 RBD IgG antibodies using a sandwich enzyme-linked immunosorbent assay (ELISA), as previously described in Trofin et al. (2022) [25 (link)]. We used a 1:10 dilution in the sample diluent provided by the kit. The samples were processed in accordance to the manufacturer’s indications (TestLine Clinical Diagnostics, Czech Republic, Catalog number: CoRG96-EIA COVID-19 RBD IgG, test specificity of 99.15% and test sensitivity of 99.9%). The optical densities were measured with a TECAN Infinite 200 photometer at 450 nm wavelength, and the results were processed with the Magellan software.
Ten cytokines and chemokines were simultaneously measured from the same breastmilk sample using a bead-based immunoassay with the Luminex Multiplexing Assay. We followed the manufacturer’s instructions (Human Premixed Multi-Analyte Kit, R&D systems, Minneapolis, MN, USA, Catalog number: LXSHM-10). The samples were analyzed using a Luminex 200 (USA) device. We quantified the following cytokines: TNF-α, IL-6, IFN-β, IL-10, IL-1β, IFN-γ, IL-2, GM-CSF, IL-5 and IP-10.
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9

Multiplex Cytokine Quantification Protocol

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The concentrations of the serum cytokines (IFN-γ, TNF-α, IL-2, IL-4, IL-6, IL-10, IL-9, and IL-17) were determined by xMAP multiplex technology [17 (link)] on the Luminex 100 system (Luminex, Austin, TX, USA), using a Human Premixed Multi-Analyte Kit (R&D Systems, Inc., Minneapolis, USA), and further analyzed using Bioplex Manager Software (Bio-Rad Life Sciences, CA, USA). All analyses were carried out in compliance with the manufacturers' instructions. Standard curves were generated for each cytokine, and the mean fluorescence intensity (MFI) of each cytokine in each well was converted into a concentration using the linear portion of the standard.
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10

Nasal Lavage Cytokine Analysis

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Nasal lavages were performed using the head‐back technique.16, 17 Briefly, using a pipette, 10 mL isotonic saline was instilled in the nasal cavity (5 mL per nostril) with the neck extended and the soft palate voluntarily closed. The head was then moved forward, allowing the lavage fluid to be blown out of the nose and into a container for further transfer to a test tube. The samples were centrifuged and frozen for later analysis as one batch.
Through magnetic Luminex assay, samples were analysed for interleukin‐6 (IL‐6), interleukin‐8 (IL‐8), interferon‐γ (IFN‐γ), tumor necrosis factor‐α (TNF‐α), myeloperoxidase (MPO) by a human premixed multi‐analyte kit (R&D Systems, Minneapolis, MN) and the Bio‐Plex 200 system (Bio‐Rad Lab, Hercules, CA). The analysis was performed in duplicates. To keep data quality high, coefficient of variance (CV) filtration was performed and duplicates exceeding a 20% CV‐cut off were omitted. Extrapolated values, as suggested by the machine, were used in case readouts were out of the detection range of the assay.
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