Msd 5 plex

Supernatant from macrophage cultures was harvested and levels of cytokines (IFN-γ, TNF-α, IL-12p70 and IL-6) assessed by MSD V-Plex assay (Meso scale discovery) according to the manufacturer’s instructions. Type I Interferons IFN-α and IFN-β were measured by ELISA kit (PBL assays science).

Human cytokine quantification was performed using a custom 30-plex Luminex panel (Life Technologies, Carlsbad, CA) containing the following analytes: IL-1RA, FGF-Basic, MCP-1, G-CSF, IFN-γ, IL-12, IL-13, IL-7, GM-CSF, TNF-α, IL-1β, IL-2, IL-4, IL-5, IL-6, IFN-α, IL-15, IL-10, MIP-1α, IL-17, IL-8, EGF, HGF, VEGF, MIG, RANTES, Eotaxin, MIP-1β, IP-10, IL-2Ra. Cell culture supernatants were flash frozen on dry ice, and thawed at time of cytokine analysis. Assays were established per manufacturer recommendations. Data were acquired on a FlexMAP 3D quantification instrument, and analysis was done using xPONENT software. Data quality was determined by ensuring the standard curve for each analyte had a 5P R² value > 0.95 with or without minor fitting using xPONENT software. To pass assay technical quality control, the results for two controls in the kit were required to be within the 95% confidence interval provided by the vendor for >25 of the tested analytes. Data were analyzed using Omega Data Analysis software. For analysis of CD22–65 CAR-T cells, culture supernatant was assayed for cytokines using the MSD V-plex (Meso Scale Diagnostics).