Fam flica caspase 1 kit

Monocytes were stimulated as described above and stained with the FAM FLICA‐Caspase‐1 Kit (Bio‐Rad ICT098), at the indicated time points, following manufacturer's instructions. Cells were extemporaneously analyzed on a Canto II cytometer. Annexin‐V staining was performed using the Annexin‐V Apoptosis Detection Kit (Invitrogen, 88‐8005‐74). Cells were then stained with propidium iodide (5 μg/ml) before acquisition on a CantoII cytometer. To obtain the response induced by the treatment, values from untreated samples were subtracted from values obtained following treatment. The sum of Annexin‐V⁺ and PI⁺ cells defined the total dead cells. The ratio was calculated as the % of Annexin‐V⁺/PI⁻ over total dead cells.

The following antibodies were used: CD45-PB, CD14-APC (both from Beckman Coulter, Brea, CA, USA, clones J33 and RMO52, respectively), and anti-ASC-PE (from Biolegend, San Diego, CA, USA, clones HASC-71). The following reagents were used: phosphate-buffered saline (PBS) (Eurobio Scientific, Luxembourg, Luxembourg), RPMI HEPES (Eurobio, Les Ulis, France), a FAM FLICA™ Caspase-1 Kit (Biorad, Hercules, CA, USA), versalyse (Beckman Coulter), nigericin (InvivoGen, San Diego, CA, USA), water for injection (Aguettant, Lyon, France), and a Cytofix/Cytoperm™ Fixation/Permeabilization Kit, stain buffer (both from BD Bioscience, Franklin Lakes, NJ, USA).

Monocytes, cultured or not for 30 min with nigericin, were washed and resuspended in RPMI 10% FBS with FLICA substrate (BioRad FAM-FLICA Caspase-1 kit), and cultured for 1 h at 37° C. Cells were then washed twice with 1X Apoptosis Buffer (from the kit) and fixed with 1x Fixative (from the kit). Cells were kept at 4°C until further staining and analysis.

Monocytes from randomly selected aSAH patients at day 1 (n = 5) and NC (n = 4) obtained as described above were used for NLRP3 inhibition assays. A total of 0.5 × 10⁶ monocytes per well were cultured in M6 plates and treated or not with 5 μM MCC-950 (INH-MCC, Ibian Technologies S.L). Cells were incubated at 37 °C and 5% CO₂ for 16 h and then both monocytes and supernatants were collected for further analysis. Monocytes were washed two times with PBS and then treated following a standard protocol using the Transcription Factor Buffer Set (Becton–Dickinson Biosciences). Cells were labeled (30 min, 4 °C) with the anti-CD14, anti-NLRP3, and anti-ASC antibodies detailed in Table e1. Additionally, caspase-1 activity was detected using the FAM FLICA caspase-1 kit following the manufacturer’s protocol (Bio-Rad Laboratories, Inc.). Cells were acquired using BD FACS-Calibur flow cytometer (Becton–Dickinson Biosciences), and data were analyzed using FlowJo vX.0.7 software (FlowJo). Gating schemes are showed at Fig. e1. Supernatants were stored at − 80 °C until analysis by ELISA (IL-18, GSDMD and TF) according to manufactures’ instructions as available in Table e2 or BD Human Inflammatory Cytokine CBA kit (IL-1β), as previously described.

bMDM were prepared and infected with both M. bovis strains as described above at an MOI of 5. Cells were harvested 24 hpi using TrypLE Express (Thermo Fisher), and inflammasome activity was investigated by measuring active CASP1 using the FAM FLICA caspase 1 kit (Bio-Rad) by following the manufacturer's instructions. bMDM were fixed with 2% PFA for 30 min before the amount of fluorescent staining was measured by flow cytometry analysis.

Keratinocytes were seeded on 8-chamber polystyrene slides (Corning Incorporated, NY 14831, USA) and were differentiated using 1.2 mM CaCl₂ overnight. Active caspase-1 was visualized using FAM FLICA Caspase-1 Kit (Bio-Rad Laboratories GmbH, D-85622 Feldkirchen, Germany). Hoechst was performed using 0.5% Hoechst reagent followed by propidium iodide (PI) staining (0.5% PI). Then, cells were fixed using fixative solution diluted 1 : 10 in apoptosis wash buffer. Pictures were taken using a Zeiss LSM 800 confocal laser scanning microscope with Zeiss ZEN 2.3 (blue edition) Software (Carl Zeiss Microscopy GmbH, Jena, Germany).