Chapter 3 │ Page 77 Slides were incubated overnight (4°C) with a primary, monoclonal antibody against ecto-CRT (ab92516, Abcam), HSP70 (ab181606, Abcam) or HSP90 (ab13492, Abcam), or the matching isotype control. The subsequent day, samples were washed and stained (room temperature, 1 hour, in the dark) with Alexa Fluor® 488conjugated Goat Anti-Rabbit secondary antibody for CRT and HSP70 (ab150077, Abcam), and Alexa Fluor® 594 Donkey Anti-Mouse secondary antibody for HSP90 (A-21203, ThermoFisher Scientific). Hereafter, slides were mounted with VECTASHIELD® Antifade mounting medium (H-1200, VECTOR laboratories) and sealed with a cover slip. Images were taken with the Olympus BX51 fluorescence microscope (WS-BX51-0169, Olympus Life Sciences). The ImageJ program was used to evaluate fluorescence intensities. 2.6. Assessment of Active Cellular ATP Secretion Besides membrane-presented markers, several immunogenic factors are secreted and/or released in the supernatant during the ICD process, including the small metabolite ATP[18]. The release of ATP from treated HNSCC spheroids was quantified using the ENLITEN® ATP Assay System Bioluminescence Detection Kit (FF2000, Promega). For this, spheroids were seeded as described before, in culture medium supplemented with heat-inactivated FBS (ThermoFisher Scientific) and treated with our NTP-CDDP combination. Supernatant was collected at 4 hours and 24 hours post-therapy, centrifuged to eliminate debris, and used immediately. Samples were pooled per condition per time point for read-outs. The assay was performed according to the manufacturer's instructions and the bioluminescence output was measured using the Spark® Cyto (Tecan). A blank (medium) control was subtracted from all samples, and the amount of ATP (moles) was determined using the standard curve equation.
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