V plex

Chemokine and cytokine levels were measured using an Electro-Chemo-Luminescence (ECL)-based assay (v-plex®, MesoScaleDiscovery). Noradrenaline levels were measured by ELISA (DLD Diagnostika) following manufacturer instructions. For corticosterone levels, blood samples were collected by retro-orbital collection 1–2 h before lights were switched off. Blood was drawn within 3 min after opening the grid with minimal manipulation prior to sampling and corticosterone was measured by ELISA kit (Enzo Life Sciences).

Interferon-γ (IFN-γ), IL-2 and tumor necrosis factor-α (TNF-α) concentrations were measured using multiplex cytokine arrays [V-PLEX, Meso Scale Discovery (MSD), Meso Scale Diagnostics, Rockville, MD] according to the manufacturer’s instructions. Assays were performed by the Human Immune Monitoring Shared Resource, University of Colorado School of Medicine, Aurora, CO. Briefly, thawed and washed T-cells were plated at 2 × 10⁵ viable cells/well in 96-U well plates and incubated for 6 h in a 37 °C incubator with 5% CO₂ prior to addition of Dynabeads (anti-CD3/anti-CD28; 2 µL/well). Cells were stimulated with beads for 18 h, then supernatants were harvested and stored at − 80 °C until assay. All assays included the T-cell comparator control described above for flow cytometry assays.
Pre-coated V-PLEX plates (MSD) were washed using an automated plate washer (BioTek ELX5012), and calibrators or thawed, diluted supernatants were added, followed by further incubation for 2 h at RT on a Compact Digital Microplate shaker (Thermo Fisher Scientific) at 600 rpm. Plates were again washed, and detection antibodies were added and incubated for 2 h at RT. After washing, 2× Read Buffer (MSD) was added and plates were immediately read on a MesoQuickPlex SQ120 electrochemiluminescent plate reader. Data were analyzed using Workbench software (MSD).