HEK293T cells transduced to express NLRP3-mNeonGreen and STING-HA were plated in 24-well glass-bottom plates (Greiner Bio-One) and, after 24 hours, stimulated with 2 µM nigericin or 1 µM diABZI with or without the addition of 10 µM C53 for 1 hour. Cells were then fixed with 2% Paraformaldehyde (Electron Microscopy Sciences) in PHEM buffer (Electron Microscopy Sciences) for 30 minutes at 37°C, washed three times with PBS and quenched with freshly prepared 0.1M Glycine for 10 minutes. Cells were permeabilized in 100% methanol for 30 minutes and stained with anti-HA (Millipore, #11867423001) and anti p-STING (Cell Signaling Technology cat. #19781s) for 1 hour at room temperature in 3% BSA, washed 5 times, and then stained with Alexa 647 anti-rat IgG (H+L) (Thermo, A-21247) and Alexa 555 plus anti-rabbit (Thermo, A32732) in 3% BSA for 1 hour. After five washes, cells were incubated in 2X SSC with 200 ng/mL DAPI (Thermo Fisher) and imaged using the Nikon microscope used for organelle pH images. Images were acquired using a 60X 1.40 NA Plan Apo λ oil immersion objective (Nikon MRD01605) with Nikon type F immersion oil with the following lasers and filters: DAPI (405 nm laser, Chroma ET455/50), NLRP3 mNeonGreen (488nm laser, Chroma ET525/36) pSTING (561 nm laser, Chroma ET605/52), and STING-HA (640 nm laser, Chroma ET705/72), assaying five z planes per field of view with 0.625 µm spacing. Fields of view were selected using NIS Elements software coordinates without manual preselection. Images are maximum projections of multiple z-stacks. Channels are: DAPI, NLRP3 mNeonGreen, pSTING, and STING.