Anti asc tms1

S. aureus was labeled with Calcein Blue, AM (5 μM, Thermo Fisher Scientific) as previously described (Cohen et al., 2016 ). BMDMs or primary human monocytes were treated as described above. In select experiments, mitochondria were labeled with Mitotracker CMXRos (Molecular Probes) prior to incubation with bacteria. The cells were incubated with labeled S. aureus at MOI 1 in a 96-well plate. For caspase-1 imaging in cells, FAM-FLICA (Immunochemistry Technologies) was added to each well 1 hr post bacterial inoculation, and cells were stained in accordance with the manufacturer’s instructions. The cells were washed 3 times in fresh PBS prior to imaging, and 20 μL cell suspension was loaded onto a coverslip. The cells were maintained at 37°C and 5% CO₂ throughout the imaging process. To image ASC, cells were allowed to adhere to coverslips overnight, prior to labeling mitochondria and infecting as described above. Following a 1-hr incubation with bacteria, cells were fixed with 10% formalin (10 min), permeabilized with 0.5% Triton X-100 in PBS (10 min), blocked with BlockAid (Thermo Fisher Scientific, 1 hr), and stained overnight with anti-ASC/TMS1 (Novus Biologicals). ASC/TSM1 was visualized with AF647 conjugated anti-rabbit IgG secondary (Molecular Probes). The slides were imaged using a Leica TCS SP5 X confocal microscope (Leica Microsystems).