Nucgreen dead 488 reagent

In order to measure caspase-1, -3, and -7 activities, FLICA 660 caspase-1 and FLICA 660 caspase-3/7 fluorometric assay (Immunochemistry Technologies, Davis, CA) was performed according to the manufacturer's protocol. HD11 cells (8 × 10⁵/well) were seeded in 6-well tissue culture plates (Cellstar, Greiner Bio-One GmbH, Kremsmunster, Austria) 24 h before infection. Cells were infected with bacteria at an MOI 1:25. After 6 h and 24 h postinfection, cells were washed 3 times with PBS, detached by EDTA-trypsin, washed again with PBS, and incubated with APC-YVAD-FMK peptide to determine the activity of caspase-1 or APC-DEVD-FMK peptide to determine the activity of caspase-3 for 30 min at 37°C. Dead cells were detected with NucGreen Dead 488 reagent (Thermo Fisher Scientific) after incubation for another 30 min. The cells were then washed and subjected to FACS analysis. Fluorescence was measured in FL-1 on the BD FACS Lyric (Becton Dickinson). A total of 10,000 events were recorded. Data were processed and analyzed using Flow Jo v10 software.

After washing the cells with warm PBS three times for 5 min, the cells were fixed with 3.7% paraformaldehyde (104005, Merck Millipore, Darmstadt, Germany). For permeabilization of the cells, we used 1% Triton X-100 (Merck Millipore, Burlington, MA, USA) for 10 min at room temperature, followed by blocking in 10% normal goat serum (NGS, Thermofisher Scientific, Bleiswijk, The Netherlands) in PBS for 15 min at RT. The cells were incubated with the primary antibody in blocking solution ( $\beta$ -tubulin III 1:200, (Sigma Aldrich)) overnight at 4 °C. After three times for 5 min PBS washing steps, the cells were exposed to secondary antibodies (anti-mouse IgG (H + L) Alexa 647 1:200, Thermofisher Scientific, Bleiswijk, The Netherlands) and 2 drops/mL NucGreen® Dead 488 reagent (R37109, Thermo Fisher Scientific)) for 3 h at room temperature. Images were obtained with a Leica TCS SP5X confocal laser scanning microscope (Leica TCS SP5X, Leica, Milton Keynes, UK). The images were built up by using proper excitation wavelengths for the different applications.