U plex assay

IL-17A, IL-17A/F, IL-17B, IL-17C, and IL-17E were measured in control sera (n = 46), BP sera (n = 83), and BP BF (n = 31) using a U-PLEX assay (MesoScale Diagnostics; Rockville; USA). U-PLEX technology allows multiplex measurement of up to 10 cytokines within a single well in a volume of 50 μL. This technique is based on electro-chemiluminescence detection. Briefly biotinylated capture antibodies were coupled to U-PLEX linkers. The U-PLEX linkers then self-assembled onto unique spots on the U-PLEX plate. After binding to the capture antibodies, detection antibodies conjugated with electro-chemiluminescent labels (MSD GOLD SULFO-TAG) bound to the analytes to complete the sandwich immunoassay. The plate was then placed into an MSD instrument (SECTOR S6000 plate reader) to acquire data. Data analysis was performed by using MSD Workbench software. Limits of detection (LLOD) were 1.6 pg/ml for IL-17A, 3.0 pg/ml for IL-17A/F, 1.0 pg/ml for IL-17B, 3.0 pg/ml for IL-17C, and 0.76 pg/ml for IL-17E.

Plasma was thawed on ice and levels of TNFα in plasma and quantified using the U-PLEX Assay (Meso Scale Discovery, Rockville, MD). The QuickPlex SQ 120 Instrument was used to evaluate cytokine levels (Meso Scale Discovery).

Blood samples were collected into EDTA-containing collection tubes from each patient within 3 days before the beginning of radiotherapy, and from 17 patients in this cohort at the end of radiotherapy. Blood samples were centrifuged within 1 h of collection at 1000 × g for 20 min at 4°C. The supernatant was collected as plasma and was stored at -80°C until analysis.
Interleukin-2 (IL-2), IL-10, IL-12p70, vascular endothelial growth factor (VEGF), tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), chemokine (C-X-C motif) ligand 3 (CXCL3), chemokine (C-C motif) ligand 5 (CCL5), colony-stimulating factor (M-CSF), granulocyte colony-stimulating factor (G-CSF), thrombopoietin (TPO), erythropoietin (EPO), WNT3A, soluble programmed death-ligand 1 (sPD-L1), interferon gamma (IFN-γ), and intercellular adhesion molecule 1 (ICAM-1) were measured by Luminex assay (R&D Systems, Minneapolis, MN). Eotaxin, FMS-related tyrosine kinase 3 ligand (FLT3L), granulocyte-macrophage colony stimulating factor (GM-CSF), IL-1α, IL-1β, IL-6, IL-8, IL-13, IP-10, and tumor necrosis factor-alpha (TNF-α) were measured by U-PLEX assay (Meso Scale Diagnostics, Rockville, MD, USA).

Cytokine and chemokine concentrations of interferon-γ (IFN-γ), interleukin-1β (IL-1β), IL-6, keratinocyte-derived chemokine (KC), tumor necrosis factor-α (TNF-α), granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-13, IL-17A, IL-17F, IL-25, IL-33, macrophage inflammatory protein-2 (MIP-2) and monocyte chemoattractant protein-1 (MCP-1) were measured in BAL fluid using a U-plex Assay (Meso Scale Diagnostics, Maryland, USA). Measurements were performed according manufacturers’ instructions. Detection limits were 0.27 pg/ml, 1.15 pg/ml, 8.40 pg/ml, 0.64 pg/ml, 0.38 pg/ml, 0.23 pg/ml, 16.60 pg/ml, 0.14 pg/ml, 14.70 pg/ml, 1.04 pg/ml, 0.23 pg/ml, 0.44 pg/ml and 2.48 pg/ml respectively.

The analysis of inflammatory markers has been described previously.^{25 (link)} In brief, a U-PLEX assay based on an electrochemiluminescent detection method (Meso Scale Diagnostics, Rockville, MD) was used to analyze the concentrations of 71 cytokines in plasma samples from patients (n = 13) with peripheral NeuP and healthy controls (n = 13). A MESO QUICKPLEX SQ 120 instrument equipped with DISCOVERY WORKBENCH data analysis software (Meso Scale Diagnostics, Rockville, MD) was used to collect and analyze data. Electrochemiluminescent signals from calibrators were fitted to a weighted 4-parametric logistic model to form standard curves.

To evaluate the levels of plasma cytokines in tumor-bearing NSG mice receiving CAR-T therapy, we used the U-PLEX assay (Meso Scale MULTI-ARRAY Technology) commercially available by MSD (Meso Scale Discovery Rockville, MD, United States). Using this platform, a cytokine panel was screened: TGFβ1, TGFβ2, IFN-α2a, IFNβ, IFNγ, TNFα, TRAIL, VEGF, IL-6, IL-9, IL-15, IL-21, IL-22, and IL-29. 25 μL of plasma from each donor was used following the manufacturer’s instructions. Electrochemiluminescence was detected using MESO QuickPlex SQ 120 (Meso Scale Discovery Rockville, MD, United States). The results were extrapolated from the standard curve from each specific analyte and plotted in pg/mL using the DISCOVERY WORKBENCH v4.0 software (Meso Scale Discovery, Rockville, MD, United States).