V plex multi spot assay system

Non-human primate cytokine/chemokine/inflammatory panel V-PLEX Multi-Spot assay system (Meso Scale Discovery, Rockville, MD) was used to quantify 24 cytokines and chemokines from plasma obtained from dams and cord blood. The assays were performed according to the manufacturer’s instructions. Briefly, plasma samples were diluted 2-fold (inflammatory, cytokine panel) and 4-fold (chemokine panel) in respective diluents and added in duplicates to plates for each panel and incubated overnight on a shaker at 4°C. Plates were washed 3 times and detection antibody cocktail specific for each panel was added to the respective plates and incubated for 2 hours at room temperature on a shaker. Plates were washed and read using an MSD instrument and the final data was obtained using the MSD discovery workbench software.

AR-BP scaffolds seeded on either the fibrous or serous side with eGFP-hAEC (71, 142, 284 cells/mm²) scaffolds were cultured in EGM-2F media, with media changed every day (n = 7 per group, per seeding concentration). After 48 and 96 h, supernatant media was collected and stored in −80 °C. AlmarBlue assay was performed at each supernatant collection timepoint and data used for normalization in subsequent experiments.
Collected media from each scaffold (n = 7 per group, per seeding concentration) was assayed using V-PLEX MULTI-SPOT assay system (Meso Scale Diagnostics, Rockville, MD) according to the manufacturer’s instructions. Assays were performed for IL-6, IL-8 and TNF-α in the pro-inflammatory panel, human GM-CSF and human IL-1α in the cytokines panel, human MCP-1 in the chemokines panel and human VCAM-1 in the human vascular injury panel. Array data were measured on a MSD QuickPlex SQ 120 (Meso Scale Diagnostics, Rockville, MD) and analyzed by MSD discovery workbench 4.0.12. Biomarkers concentrations were determined by comparing data to standards obtained with test kits. Obtained concentrations were normalized to AlamarBlue data obtained following supernatant collection.