24 mm glass coverslips

Monocytes, plated on 24 mm glass coverslips (Marienfeld, Germany), were incubated at 37 °C in the presence of LPS or LRZ for 6 h (N = 6). Samples were then washed with PBS, fixed and permeabilized with 4% paraformaldehyde and 0.5% TritonX-100 in PBS. After washing and saturation with PBS supplemented with 2% (w/v) BSA (Sigma), cells were labeled with primary antibodies against IL-1β (mAb 3ZD) and LAMP2A (rabbit, Abcam, UK) in PBS/BSA, followed by incubation with specific secondary antibodies labeled with Alexa-546 (Life Technologies, USA) or Alexa-647 (Invitrogen, USA). Images were obtained using a Leica TCS SP8 scanning confocal microscope equipped with a 63 × /1.4 oil objective and LAS AF software with a Z-stack of 170 nm. Reconstructed 3D volume was obtained using ImageJ. For TIRF analyses, cells were examined under a Leica SR Ground State Depletion (GSD), microscope, with a 160 × /1.45 objective and an sCMOS camera (Hamamatsu Flash ORCA 4.0). In order to enhance the signal vs. background and identify/quantify the vesicles, images were filtered with a Laplacian of Gaussian filter (using the FeatureJ-Laplacian plugin of ImageJ with a smoothing scale parameter of 1), before applying a histogram based threshold.

Peripheral blood mononuclear cells were plated at 5 × 10⁶/well in 24 mm glass coverslips (Marienfeld, Germany) for 30 min, washed in PBS and incubated in complete medium at 37 °C with different stimuli. Time-lapse photographs were then taken every 3 min for up to 6 h using a Zeiss Axiovert S100 TV2 microscope (Zeiss) equipped with a 63 × /1.4 lens and a Hamamatsu OrcaII-ER camera (Hamamatsu City). Images were analyzed using ImageJ or Oko-vision (Okolab) softwares^{54 (link)}. Monocytes stimulated for 6 h with LPS or LRZ or for 3 h with LPS and then with R848 and zymosan for 3 h were stained for 10 min with PI (5μg/ml) and images were recorded with the software LASX on a Leica DMI6000 microscope (Leica Microsystems, Mannheim, Germany) with a 63 × /1.45 objective and a sCMOS camera (Hamamatsu Flash ORCA 4.0).