Magpix system

MILLIPLEX MAP Human Cytokine/Chemokine Magnetic Bead Panel kit (HCYTOMAG60, Merck Millipore, Darmstadt, Alemania) was used to measure the cytokines levels from cell culture supernatant in THP-1 and Mo cells stimulated with rND1, according to the manufacturer’s instructions, in a MAGPIX^® System (Merck, Darmstadt, Germany). All samples were run in duplicate, and basal culture medium was used as blank. The cytokines included were EGF, Eotaxin, G-CSF, GM-CSF, IFNa2, IFNy, IL-10, IL12p40, IL12p70, IL13, IL15, IL17, IL1ra, IL1α, IL1β, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IP10, MCP1, MIP1α, MIP1β, TNFα, TNFβ, and VEGF. The concentration values were obtained from the mean fluorescent intensity (MFI) by using platform MAGPIX^® (software Milliplex Analyst v 3.5.5.0). For each target, a 5-point standard curve was generated from the reference cytokines pool.
To reduce the risk of parallel pathways with pro- and anti-inflammatory downstream effectors during in vitro studies, we analyzed by Luminex the cytokine composition of the cell culture medium supplemented with 1% FBS (Supplementary Materials, Table S4).

Protein was extracted using RIPA buffer with 1× protease inhibitors, 2 mM PMSF and 1 mM sodium orthovanadate (Sigma-Aldrich) was added to cells and incubated on ice for 20 min. Following centrifugation at 10,000 rpm for 10 min at 4°C, the resulting lysate was stored at −80°C. Protein quantification was performed using the bicinchoninic acid (BCA) assay (Pierce).
Magnetic bead assays were performed on the Luminex® MagPix® System (Merck Millipore, 80–073) using Milliplex Map Phospho Mitogensis RTK Magnetic Bead 7-Plex kit (Merck Millipore, 48–672 Mag) and Milliplex Map phosphor Human SRc Family Kinase Magnetic Bead 8-Plex kit (Merck Millipore 48-650Mag).
Protein (1–10 μg) was diluted in appropriate volume of assay buffer (final volume: 25 μL/well) and the assay was performed as per the manufacturer’s instructions.

We collected the following laboratory tests upon study inclusion: blood count, C-reactive protein, platelets, creatinine, blood urea nitrogen, bilirubin, inflammatory biomarkers (IL-1B, IL-4, IL-10, INF-g and TNF-α and neuronal injury biomarkers (S100B, Neuron Specific Enolase and Tau protein).
Three registered nurses performed the sampling while patients were in the ED, which consisted of 30 ml of blood collected by venipuncture. Blood samples used for brain injury biomarkers analysis were immediately centrifuged for 10 min, and plasma was preserved at − 20 °C for up to 48 h before being transferred to a − 80 °C freezer for long-term storage and further processing.
We measured cytokine plasma levels using the magnetic bead immunoassay Milliplex® and the MAGPIX® System (Merck Millipore, USA).
The sampling procedures were performed on inclusion (S1) and repeated 72 h after inclusion (S2)^{21 (link)}. Participants who were discharged or died within 72 h of admission, or refused to provide additional samples, were not punctured again. We obtained a third sample (S3) from participants who converted either from a negative to positive CAM (incident delirium) or from a positive to negative CAM (delirium resolution) after S2 (Fig. 1).Figure 1

Flowchart of study procedures.

The plasma protein concentrations of ADAMTS13, eotaxin, Fas, GDF15, GROα, ICAM1, IL1α, IL6, IL7, IL8, IL10, IL15, MCP1, MDC, MIP1α, MIP1β, MMP1, MMP7, MMP9, MPO, OPN, PAI1, PARC, RAGE, RANTES, SOST, TARC, TNFR1, TNFR2, TNFα, and VEGFA were quantified using commercially available multiplex magnetic bead-based immunoassays (R&D Systems) on the Luminex xMAP multianalyte profiling platform and analyzed on MAGPIX System (Merck Millipore). Activin A concentration was determined by a Quantikine ELISA Kit (R&D Systems). Protein names, abbreviations, and aliases are provided in Additional file 1: Table S1. All assays were performed according to the manufacturer’s protocols. In instances where a biomarker was below the limit of detection in a participant sample, a value of half of the lowest measured value for that analyte was assigned.

Blood samples were obtained by cardiac puncture right before transcardial perfusion to determine circulating cytokine levels 24 h after stroke. Plasma was prepared from blood by adding 50 μL/mL 0.5 M EDTA and samples were stored at -80°C until used. Cytokine detection was carried out with a custom-made rat cytokine/chemokine magnetic bead Milliplex MAP panel (Millipore, Temecula, CA, United States) following the manufacturer instructions. A 96-well plate was used to simultaneously determine the concentrations of IL-1β, IL-4, IL-6, IL-10, IL-12p70, IL-17, TNF-α, CCL11, INFγ, CX3CL1, MIP-1α, and VEGF. The plate was read on a MAGPIX system (EMD Millipore, Darmstadt, Germany). Results were analyzed using the xPONENT software (Luminex, Madison, WI, United States). Standards of 200–16,000 pg/mL were used for generating the corresponding concentration curve of each analyte. Determinations were done in duplicates per biological replicate (n = 3).