Magpix system

Interleukin-6, G-CSF, CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP-1β), CCL5 (RANTES), CXCL2 (MIP-2), and CXCL9 (MIG) levels in ex vivo supernatants were determined using a liquid multiarray system (Bio-Plex Pro™) according to the manufacturer’s instructions. Commercial multiplex-coated beads (custom-made cytokine panel), biotinylated Abs, and Beadlyte microtiter 96-well filter plates were obtained from Bio-Rad. Data were collected with Bio-Plex Manager™ software and analyzed with Bio-Plex^® MAGPIX system. Standard curves for each cytokine and chemokine were obtained using the reference concentrations supplied by the manufacturer.

Multiplex analysis was performed using a Bio-Plex MAGPIX system (#171015044) and Bio-Plex Pro-Wash Station (Biorad). All cell lysates were prepared using standard cell lysis buffer (50 mM Tris HCl pH7.4, 150 mM NaCl, 1 mM EDTA, 1% (v/v) TritonX-100) supplemented with protease and phosphatase inhibitors. Lysates were analysed on all kits, according to manufacturer’s instructions. Data was generated using Bio-Plex Manager MP and analysed on the Bio-Plex Manager 6.1 software.
Lysates were analysed using the Milliplex map DNA Damage/Genotoxicity Magnetic Bead Panel (7-plex), Milliplex map Early Apoptosis Magnetic Bead (7-plex), Milliplex map TGFβ Signalling Pathway Magnetic Bead kit (6-plex), Bio-Plex Pro Cell Signalling Akt Panel (8-plex), Bio-Plex Pro Cell Signalling MAPK Panel (9-plex), Bio-Plex Pro RBM Apoptosis Panel two and Bio-Plex Pro RBM Apoptosis Panel 3. Individual beads were also used to analyse NF-κB (Ser536), IκBα (Ser32/36), c-Jun (Ser63), total P53 and cleaved PARP (Biorad). The data was normalized to the median value at the 0 hr time point for each analyte and a log transformation was conducted on the resulting dataset. The principal component analysis was performed using MATLAB and Statistics Toolbox Release 2019a (The Mathworks, Inc, Natick, Massachusetts, United States of America).

To explore the potential mechanism by which patients exist cognitive impairment, Luminex liquid suspension chip detection was performed to compare the differential expression of 48 cytokines including pro-inflammatory, chemokines, and growth factors (Supplementary Table 1).
All peripheral blood samples were collected between 8:00 to 12:00 by using anticoagulant tubes and the plasma was isolated by centrifugation (3000 rpm for 15 min at 4℃). The separated plasma was deposited at -80 ℃ until being used. According to the manufacturer's instructions using the Bio-Plex MagPix System, the plasma concentration of the 48 cytokines were simultaneously measured by the Bio-Plex Pro Human Cytokine Screening 48-Plex Panel (Bio-Rad, Hercules, CA, USA), and streamlined data were analyzed with the Bio-Plex Manager (Luminex, Austin, TX, USA), agented by Wayen Biotechnologies (Shanghai, China). The calculation of the concentration was based on the cytokine’s fluorescence values which resulted from the standard substance included in the 96-well plate. The concentration below or above the limit of the standard concentration could not be detected and treated as missing data. All the results are presented as picograms per milliliter.

Cytokine levels in 54 sinus washes (stored frozen at −80°C) from 22 people were quantified using a Luminex MAGPIX system with either a Bio-Plex Pro human inflammation 24-Plex panel or a Bio-Plex Pro human Th17 cytokine panel 15-Plex panel (Bio-Rad, Hercules, CA, USA). Cytokines were omitted from further analyses if their concentration was close to the lower limit of detection in most samples, based on manufacturer-specified values and examination of the 5-parameter logistic (5pl) standard curves produced by the Bio-Plex Pro software. Seven cytokines were included in the predictor/responder analyses in Fig. 5. All cytokine concentrations were log transformed, except pentraxin 3 (PTX3), which was sufficiently normally distributed according to the Shapiro-Wilks test, without additional log transformation.

Cell supernatants were collected from M0, M1, M2a, and M2c macrophages cultured either in hPL or FBS on day 7. The supernatants were centrifuged at 500g for 5 min and then stored at −80 °C. A multiplex bead-based immunofluorescent assay (R&D Human Magnetic Luminex Customized Assay, R&D Systems Inc., Minneapolis, MN, USA) was used to determine the protein secretion profiles of the macrophages. For the analysis, cell supernatants were thawed and incubated with a cocktail of antibodies pre-coated onto magnetic microparticles. A cocktail of biotinylated antibodies was added, followed by streptavidin–phycoerythrin conjugate. Finally, the microparticles were read using a MagPix™ System (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Samples were analyzed in duplicate. A 5-parametric standard curve (Bio-PlexManager software, version 6.1.1, Biorad) was used to determine the protein concentration of samples. For each analyte, data were subtracted from background values measured in hPL-based or FBS-based medium and maintained at 37 °C for 2 days in the absence of cells. Additionally, the residual values of IL4, IL13, and IL10 added to the medium at the same concentrations used for M2a and M2c polarization and kept at 37 °C for 2 days were subtracted from the values measured for these cytokines in the supernatants collected from the M2a and M2c macrophages.