F bottom clear cell culture treated 96 wells plates

All robotic steps were performed on a Tecan Freedom EVO platform. Compounds from the Prestwick Chemical Library® were evaluated at a 1:1,000 or 1:2,000 dilution of the original stock for plates 1–14 and plate 15, respectively (see Table S1). 1 μL of DMSO solutions was spiked into dry well of F-bottom clear cell culture treated 96-wells plates (Greiner Bio One), with columns 1 and 12 devoted to controls and used to calculate the Z′-factor. U937 cells expressing p.S242R MEFV were treated or not (counterscreen) with doxycycline (1 μg/mL) for 16 h, centrifuged and seeded at 10⁵ cells per well (100μL final volume) in RPMI 1640 without phenol red, 10% FCS, 1mM HEPES, 1% PSA, 1mM Glutamine, in the presence of propidium iodide at 5 μg/mL. Following 90 min of incubation at 37°C, fluorescence intensity (excitation wavelength at 535 nm and emission wavelength at 635 nm) corresponding to propidium iodide incorporation was measured on a microplate reader (Infinite M1000, Tecan). The average Z′ value was 0.67 ± 0.12, indicating a robust and reliable assay. Mean fluorescence + 3SD was retained as a threshold. Compounds triggering cell death only in the presence of doxycycline were considered as pyrin-specific and defined as hits.