Las x life science software

BMDMs were loaded with 1 μg/mL Acridine Orange for 20 min (Antunes et al., 2001 (link)). Images were acquired using a Leica SP8 confocal microscope (Leica Microsystems) and data processing was performed with the LAS X Life Science software (Leica Microsystems). Additional samples were acquired in a FACSCanto II equipment (BD Biosciences) and lysosomal rupture was followed by the loss of red fluorescence from the acidic lysosomal compartment in the PerCP-Cy5.5 channel. Data were analyzed by FlowJo Software (Tree Star).

THP-1-ASC-GFP cells and ASC-Citrine BMDMs were acquired in a FACSCanto II equipment (BD Biosciences, Franklin Lakes, NJ, US). Gates for priming and ASC-specks formation were established according to the relative distribution of the cell population in the FITC-W and FITC-A channels (Hoss et al., 2018 (link)). Data were analyzed by FlowJo Software version 10.8.1 (Tree Star, Ashland, OR, US). In addition, ASC-Citrine BMDMs were imaged with a SP8 confocal microscope (Leica Microsystems, Wetzlar, Germany) equipped with a 63 × /1.40 oil objective. Data processing was performed with the LAS X Life Science software (Leica Microsystems).

Cells were grown on sterile glass coverslips, fixed with 4% PFA, and washed three times with PBS. Fixed cells were permeabilized and blocked by incubation for 40 min with PBS containing 0.25% saponin and 2% FBS. Cells were incubated for 1 h with primary antibodies diluted 1/200 in PBS with saponin and FBS. The following antibodies were used: a rabbit polyclonal antibody specific for the BUNV nucleocapsid (N) protein [7 (link)], a mouse monoclonal antibody specific for BUNV Gc glycoprotein, kindly provided by Prof. Richard M. Elliott [39 (link)] and a rabbit anti-Giantin polyclonal antibody that is a marker of the Golgi complex (BioLegend, San Diego, CA, United States; 924302). Alexa Fluor–conjugated antibodies (Invitrogen) were used as secondary antibodies diluted 1/500 in PBS with 0.25% saponin and 2% FBS. Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) diluted 1/200 in PBS with 0.25% saponin and 2% FBS for 20 min. All incubations were conducted at RT. Coverslips were mounted using Prolong-Gold (Life Technologies, Carlsbad, CA, USA). Confocal microscopy images were acquired using a Leica STELLARIS 5 confocal multispectral microscope equipped with an HCX PL APO 63.0 X/1.4 NA oil objective and LAS X Life Science software (Leica Microsystems).

Immunofluorescence images were acquired with 20 × or 40 × magnification with a fluorescence microscope (DM6000B, Leica) and LAS X Life science software (Leica) using the same microscope settings (exposure time, gain, lamp intensity, magnification) for each experiment or test series. Images were assessed using the FilamentTracer algorithm of the commercially available IMARIS® software, which allows semi-automatic detection, tracing and measurement of neuronal cells and their processes. The software evaluates neurite features such as neurite area, neurite length, neurite diameter and neurite branches. We calculated neurite markers relative to the number of cell nuclei as ratio of a given neurite marker per nucleus.