Fam flica

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).

Spleens were mashed in RPMI 1640 (SIGMA) containing 1% fetal bovine serum (FBS), strained through a nylon filter, then washed before staining with FAM-FLICA according to the manufacturer’s instructions (ImmunoChemistry Technologies LLC), washed with PBS then stained with anti-F4/80 Pacific Blue antibody (MCA497PB Bio-Rad) and anti-Ly6G APC-Cy7 antibody (BD Biosciences), washed with PBS and fixed in 10% formalin then visualized by BD FACSCanto II and analyzed by Cytobank software (Cytobank Inc.)

For active caspase-1 and caspase-3/7 staining, cells were washed with PBS/2% FBS and incubated with either FAM-FLICA or FLICA 660 (ImmunoChemistry Technologies) per the manufacturer’s protocol. For fixable viability staining, cells were washed; stained with Zombie NIR, violet, or ultraviolet (BioLegend) for 15 minutes at 1:1000 dilution at room temperature; then washed with PBS/2% FBS. For antibody staining, cells were incubated with human or mouse Fc block (Miltenyi Biotec) and stained 20 minutes at 4°C. Data were acquired using a BD Symphony, Canto, or LSRII flow cytometer (Becton Dickinson). FCS files were analyzed using FlowJo v10 software (Tree Star). Relative MFIs between replicates were compared as fold-change differences rather than raw MFI values because of variation between flow cytometers.

Larvae at 6 dpf were incubated with FAM-FLICA (Immunochemistry Technologies) at 1:300 for 12 h. The next day, larvae were washed with E3 medium and fixed in PIPES/1.5% formaldehyde buffer at 4°C overnight. Larvae were washed three times with PBS 1×, and livers were carefully dissected with the use of forceps on a stereomicroscope (Leica MZ 9.5). After dissection, livers were immersed in PBS and images were acquired on a spinning disk confocal microscope (CSU-X; Yokogawa) with a confocal scanhead on a Zeiss Observer Z.1 inverted microscope equipped with a Photometrics Evolve EMCCD camera using an EC Plan-Neofluor 40×/0.75 NA M27 air objective with a 1-μm interval. Mean fluorescence intensity was measured from MIPs using Image J.

5 × 10⁵ NPCs from Snip1[+/+] and Snip1[flox/flox] embryos were seeded onto each well of matrigel-coated 6-well plates. On the following day, cells were incubated with mCherry-Cre lentivirus (Vector Core Lab at St. Jude Children’s Research Hospital) for 8 h, washed twice with 1XPBS, and cultured for 3 days. To quantify the population of cells with active caspases 3 and 7, cells were incubated at 37 °C with reconstituted FAM-FLICA® at a 1:300 dilution (ImmunoChemistry Technologies 94) for 30 min. Cells were fixed in a 4% formaldehyde solution at room temperature for 15 min and washed twice with 1X PBS. FAM-FLICA–positive cells were quantified by FACS (Excitation: 492 nm, Emission: 520 nm). FACS data were analyzed by FlowJo. To examine whether cell death is via activation of caspase 8 or 9, Z-IETD-FMK (a caspase 8 inhibitor) and Z-LEHD-FMK TFA (a caspase 9 inhibitor) were dissolved in DMSO at 50 mM (Compound Management Center at St. Jude Children’s Research Hospital). After cells were incubated with mCherry-Cre lentivirus for 8 h, these compounds were added at a series of concentrations and incubated for 3 days before FACS analysis. For all the inhibitor treatment assays, the medium with inhibitors was changed every 2 days. The gating strategy and representative FACS plots are shown in Supplementary Information (Supplementary Fig. 4e, Supplementary Fig. 14).