Hanne Verswyvel

Chapter 3 │ Page 76 fluorescence whole-well images were analyzed with the Orbits® platform. Total organoid survival area was determined by subtracting the green-labeled dead area from the brightfield organoid area, and was thereafter normalized to the initial area for each organoid individually. 2.5. Immunofluorescence Analysis of Membrane-Associated DAMPs 2.5.1. Spheroid Embedding and Preparation of Microscopy Slides Spheroids were collected 24 hours posttreatment from the 96-well ULA plates, washed twice with PBS, and then fixed overnight in 4% formaldehyde at 4°C. The next day, the fixed spheroids were transferred into in-house developed, custom-made microarray molds prepared from 4% agarose and a 3D-printed grid maker (Figure 1). These molds were freshly produced prior to spheroid transfer to ensure optimal integrity. Spheroids were sealed within the molds using preheated low-melting-point agarose. Once the gels had solidified, the microarray molds were placed in plastic histology cassettes for further processing. 2.5.2. Immunofluorescence For visualization of ICD-related markers CRT, and HSP70 and HSP90 on the tumor cell membrane, 5µm-thick, consecutive slides from the HNSCC spheroids were cut and processed together. Tumor slides were baked (60°C, 2 hours) and depara inized, whereafter heat-induced antigen retrieval with sodium citrate bu er (10mM Sodium Citrate, 0.05% Tween 20, pH 6.0) was performed (100°C, 20 minutes). Aspecific binding was prevented by blocking in serum (10%) matched with the host species of the secondary antibody. Figure 1: In-house created microarray mold. Spheroids were transferred to individual pores created in the agarose.

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