Spe confocal system

After seven days of culture chitosan-based matrices and cytochemistry control samples (glass coverslips containing cells) were fixed overnight (4 °C) with paraformaldehyde, PFA 4% (in PBS), permeabilized with 0.2% Triton-X (room temperature, 10 min) and stained at room temperature for 1 h with Alexa488 conjugated Phalloidin (1:400 in PBS) Molecular Probes), to observe cell’s actin cytoskeleton. The samples were mounted on fresh PBS on a glass slide and imaged on a Leica SPE confocal system (Wetzlar, Germany). Confocal images were analyzed using Fiji, version 1.48.

Cells were fixed with 4% formaldehyde in PBS for 20 min, permeabilized with cold (−20 °C) 70% ethanol for 20 min, blocked with 8% BSA in PBS for 1 h, incubated with the first antibody (caspase-8, 1/50 dilution; FADD, 1/100 dilution; NOX2, 1/250 dilution; p47^PHOX, 1/250 dilution) in 1% BSA in PBS for 1.5 h, then with PLUS and MINUS PLA probes (Olink Bioscience, Uppsala, Sweden) for 1 h at 37 °C, then with ligation mix (Olink Bioscience) for 30 min at 37 °C, then with amplification mix (Olink Bioscience) for 100 min at 37 °C before being mounted using Duolink In Situ Mounting Medium with DAPI (Olink Bioscience). Confocal images were acquired on a Leica SPE Confocal system, sequential acquisition using 63X/1.3 oil immersion. 3D pictures were realized with the Bitplane Imaris software v. 7.7.2.

Cells were washed in PBS, fixed with 4% formaldehyde in PBS for 20 min, washed with PBS, post-fixed and permeabilized with cold 70% ethanol for 20 min, washed with PBS, blocked with 8% bovine serum albumin (BSA) in PBS for 1 h, incubated with the first antibody (active caspase-3, 1/250; γ-H2AX, 1/500; calreticulin, 1/250; LC3, 1/250) in 1% BSA in PBS for 2 h, washed and incubated with a secondary antibody conjugated with Alexa Fluor 488 or 555 for 1 h, washed and mounted by using Vectashield mounting medium with DAPI (Vector Laboratories, Burlingame, CA, USA). Mitotracker orange (#M7510, Thermo Fisher Scientific, 100 nM) was incubated 30 min with living cells at 37 °C. FAM-DEVD-FMK and FAM-LETD-FMK detection kit FLICA (Bio-Rad, Marnes-la-Coquette, France) was used according to the manufacturer’s instructions. Confocal images were acquired on a Leica SPE Confocal system, sequential acquisition using 63X/1.3 oil immersion. For 3D pictures, we used Bitplane Imaris software v. 7.7.2. Lines intensity profiles were realized using the LAS AF software v. 2.7.3.9723. Fluorescence quantification and co-localization quantification were calculated using ImageJ 2.0.0-rc-69/1.52p.