Zen blue microscope software

THP-1 cells were seeded into 96-well plates at a concentration of 40,000 cells per 100 μL complete media. To visualize the role of RTD on inflammasome activation, cells were seeded onto 12 mm glass coverslips (Neuvitro Corporation, Vancouver, WA, USA) in a 24-well plate at a concentration of 2 × 10⁵ cells in 0.5 mL complete medium for confocal imaging. Cells were differentiated in the wells for 48 h in 100 nM PMA. The complete medium was aspirated and replaced with RPMI +1% HI-FBS before addition of LPS (100 ng/mL), RTD-1 (7.14 μg/mL), or azithromycin (50 μg/mL) for 6 h. ATP (5 mM; Millipore Sigma) was added 45 min before cells were labeled using the FAM-FLICA^® Caspase-1 Assay Kit (ImmunoChemistry Technologies, Bloomington, MN, USA) according to the manufacturer’s instructions. Inflammasome activity was measured using a Synergy H1 kinetic fluorescent microplate reader. Cells seeded onto the coverslips were fixed and mounted using a Fluoroshield Mounting Medium (Abcam, Cambridge, UK). Representative confocal images were taken using a ZEISS LSM 880 with Airyscan (ZEISS, Oberkochen, Germany). The images were analyzed using ZEN Blue Microscope software (ZEISS). Lastly, RTD-1′s direct effect on caspase-1 inhibition was assessed in vitro using a fluorogenic substrate assay according to the manufacturer’s instructions (Abcam) using a Synergy H1 kinetic fluorescent microplate reader.