Activation of NLRP3 Inflammasome in Macrophages

Bone marrow-derived macrophages were seeded in 4-chamber slides (Thermo Fisher Scientific, Waltham, MA, USA) at a density of 1 × 10⁵ cells per chamber. After an overnight incubation, the cells were primed with 50 ng/ml LPS for 3 h, treated with 10 μM InflammaProbe-1 for 1 h, and stimulated with 10 μM nigericin for another hour to induce NLRP3 activation, as described in the literature (18 (link), 33 (link)). Then, the cells were washed twice with PBS, fixed with 4% neutral buffered formalin (NBF) for about 2 min, and washed again twice with PBS. Immediately, the chambers were removed from the microscope slides in accordance with the manufacturer’s instructions. Cells were mounted with Prolong™ Diamond Antifade Mountant with DAPI (Invitrogen, Waltham, MA, USA) and imaged through confocal fluorescence microscopy using an LSM 710 inverted microscope (Zeiss™, Jena, Germany). The InflammaProbe-1 fluorescence intensity of each image was expressed as RFU per cell, which was calculated by dividing the raw integrated density, measured with Fiji ImageJ2 software, by the number of DAPI-stained nuclei present in the image. The data were representative of four replicates per group.