Msd discovery workbench software version 4

We measured the concentrations of cytokines/chemokines in the MD fluid collected from the injury site. These molecules were selected because they have been implicated in the pathophysiology of spinal cord injury (see supplement), they are <30 kDa (i.e. can cross the MD membrane) and they are part of our multiplex electrochemiluminescence assay panel. For each patient, MD vials were grouped by sampling period: 6 hours pre-cooling, 6 hours at 33 °C and 6 hours post-rewarming i.e. at 37 °C. MD vials, each containing about 10 μL, were stored at −80 °C and analyzed in a blinded fashion at Charité-Universitätsmedizin Berlin by multiplex electrochemiluminescence assays (Meso Scale Discovery, Rockville, MD, USA) using the following antibody sets from the U-PLEX Biomarker Group 1 Human Assays panel (K15067L): IL1α, IL1β, IL4, IL8, IL10, IP10, GROα, MCP1, MIP1α, MIP1β. If necessary, the MD samples were diluted to the minimum required volume for a single determination (25 µl) or analyzed directly according to the manufacturer’s protocol. 96-well plates including samples, blanks and recombinant standard concentrations were measured and unknown concentrations calculated, using the MESO QuickPlex SQ 120 Reader and the MSD DISCOVERY WORKBENCH software version 4.0 (Meso Scale Discovery, Rockville, MD, USA), respectively.

Synovial fluid (primary, n = 21; revision, n = 24) and tissue homogenates from infrapatellar fat pad (primary, n = 28 patients; revision, n = 32 patients) and synovial membrane (primary, n = 29 patients; revision, n = 32 patients) were assessed for expression of 39 protein markers using a human V-Plex electrochemiluminescence detection kit (K15209D; MesoScaleDiscovery, Rockville, MD), as per manufacturer's instructions. Analysis of results was performed using the MSD Discovery Workbench software version 4.0 (MesoScaleDiscovery, Rockville, MD).

The plasma samples were heat-inactivated for 60 min at 56 °C prior to 1:5000 dilution. eHCoV antibody levels were determined using Mesoscale Diagnostic’s V-PLEX Coronavirus Panel 2 which includes pre-fusion stabilized spike trimers from all four eHCoVs, plus SARS-CoV-1 spotted together in individual wells in a 96-well format. Binding specificity for HCoV-OC43 and SARS-CoV-2 spike has been previously determined for this commercially available assay [23 (link),24 (link)]. The assay was performed following the manufacturer’s instructions. Diluted samples, along with manufacturer-provided calibrators and controls, were applied to blocked plates and incubated for 2 h. Washed plates were incubated with detection antibody for 1 h, followed by the addition of MSD GOLD Read Buffer B. The plates were read on the MSD instrument and the raw data were processed in MSD Discovery Workbench software (version 4.0) (Mesoscale Diagnostics, Rockville, MO, USA). IgG antibody levels for each antigen were calculated in Workbench based on the calibrator standard curve fit and reported in Arbitrary Units/mL (AU/mL). IgG concentrations that were below the manufacturer’s detection or curve fit limits were set to 0.