We selected 16 inflammatory markers based on previous reports.21 (link)23 (link) The protein concentrations of tissue extracts were determined using the Pierce 660 nm Protein Assay Kit (Thermo Fisher Scientific ., Waltham, MA, USA). Enzyme-linked immunosorbent assays for IL-22 and human neutrophil elastase (HNE) were performed with commercially available kits (all from R&D Systems, Minneapolis, MN, USA). Multiplex cytokine analysis kits (IL-1β, IL-5, IL-6, IL-8, IL-17A, IFN-γ, eotaxin-3, myeloperoxidase [MPO], matrix metalloproteinase [MMP]-9, TGF-β1, and periostin) were purchased from R&D Systems, and data were collected using a Luminex 100 reader (Luminex, Austin, TX, USA). Data analysis was conducted using MasterPlex QT version 2.0 (MiraiBio, Alameda, CA, USA). The levels of total IgE, specific IgE to staphylococcal enterotoxins (SE-IgE [SEA, SEB, SEC, and TSST-1]), and eosinophil cationic protein (ECP) in nasal tissue homogenates were measured using the ImmunoCAP assay (Thermo Fisher Scientific). A SE-IgE level > 0.35 IU/mL for at least one SE-specific IgE (SEA, SEB, SEC, and TSST-1) was defined as SE-specific IgE positive. All assay procedures mentioned were run in duplicate according to the manufacturer’s protocol. All protein levels in the tissue homogenates were normalized to the concentration of total protein. Further details are provided in the Supplementary Data S1 .
Multiplex cytokine analysis kits
Manufactured by R&D Systems
Sourced in United States
Multiplex cytokine analysis kits are designed to quantitatively measure multiple cytokines simultaneously from a single sample. These kits utilize bead-based technology and flow cytometry to enable the detection and quantification of a panel of cytokines in a single experiment.
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2 protocols using multiplex cytokine analysis kits
1
Inflammatory Marker Quantification in Tissue
2
Cytokine Profiling in SJS/TEN Patients
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In January 2017, we started a series of SJS/TEN-related scientific studies, which were approved by the ethics committee of the hospital. For cytokine tests, 17 patients with SJS/TEN, 3 with acute pancreatic injuries, were enrolled from December 2017 to June 2019 after consent was obtained from each individual. Simultaneously, 18 sex- and age-matched healthy controls were recruited (Supplemental Table I; available at https://data.mendeley.com/datasets/6vycp9yvnp/draft?a=00db49dc-1212-47e3-9596-dd5ac2e136d4 ). Peripheral blood (5 mL) was obtained from each of 17 patients during the acute stage before treatment, as well as from 18 controls. Serum specimens were suspended from clotted blood centrifuged at 4°C and 2,500 rpm for 10 minutes and then stored at –80°C until tested.
Serum samples were tested for 6 cytokines; namely, tumor necrosis factor α, IL-6, IL-18, IL-15, IL-12p70, and soluble CD56. Multiplex cytokine analysis kits were obtained from R&D Systems, and data were collected with Luminex 100 (Luminex, Austin, TX). Data analysis was performed with MILLIPLEX Analyst (version 5.1, EMD Millipore Corporation, Billerica, MA).
Serum samples were tested for 6 cytokines; namely, tumor necrosis factor α, IL-6, IL-18, IL-15, IL-12p70, and soluble CD56. Multiplex cytokine analysis kits were obtained from R&D Systems, and data were collected with Luminex 100 (Luminex, Austin, TX). Data analysis was performed with MILLIPLEX Analyst (version 5.1, EMD Millipore Corporation, Billerica, MA).
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