Chapter 3 │ Page 78 2.7. Detection of Post-Mortem Cellular HMGB1 Release HMGB1 release is another hallmark associated with ICD that can be measured in the supernatant of treated cell cultures. HMGB1 concentrations in the medium were quantified with the HMGB1 express ELISA kit (30164033, Tecan). Samples were collected 48 hours and 72 hours after NTP-CDDP treatment on HNSCC spheroids, and pooled per condition at each time point. After centrifugation, the supernatant was frozen down at -80°C until further analysis. The analysis was executed following the manufacturer’s protocol, and optical density read-outs were performed directly after the final experimental step using the Spark® Cyto (Tecan). Extracellular HMGB1 levels were determined via the standard curve equation, after correcting all samples to the proposed reference wavelength (600 650 nm). A duplicate of the collected supernatant was stored at -80°C for subsequent analysis of cytokine and chemokine release following treatment (detailed in the next section). 2.8. Profiling of Cytokine and Chemokine Release Several cytokines and chemokines can be released in the immunogenic process of cell death. In this context, the supernatant of NTP-CDDP treated HNSCC spheroids was examined on the presence of several cytokines (IFN-α, IFN-β, IL-1β, IL-6, IL-8, IL-10, TNF-α and TGB-β) and chemokines (CXCL9, CXCL10, CCL4 and CCL5) using the electrochemiluminescence technology of MesoScale Discovery Inc. A U-plex custom Immuno-Oncology Group 1 multiplex panel (K151AEM-1) was used for the detection of IFN-α, IL-1β, IL-6, IL-8, IL-10, TNF-α, CXCL9, CXCL10, CCL4 and CCL5. IFN-β and TGF-β were analyzed with a S-plex (K151ADRS-1) and a U-plex (K151XWK-1) detection kit, respectively. Samples were processed according to the manufacturer’s instructions, and the electrochemiluminescence signal was measured on a SECTOR3000 plate reader (MesoScale Discovery Inc.).
RkJQdWJsaXNoZXIy MTk4NDMw