Sis item

For neutral BODIPY staining, 2 × 10⁵ RAW cells were seeded in each compartment of compartmented 35-mm sterile culture dishes with glass bottom (Cellview – Greiner). Cells were incubated for 4 h with AS03. Coverslips were washed and cells were stained with BODIPY 493/503 (Thermofisher) (1/500) and visualized with a Zeiss Axio Observer wide-field microscope.
For TEM, samples were washed with PBS and fixed with ice-cold glutaraldehyde 2% (EM grade, Sigma). Then, they were post-fixed in OsO₄ (2%) in 0.1 M cacodylate buffer (pH 7.2), serially dehydrated in increasing ethanol concentrations, embedded in Agar 100 resin (Agar Scientific Ltd, UK) and left to polymerize at 60°C for 2 days. Ultrathin sections (50–70 nm thick) were produced with a Leica EM UC6 ultra-microtome, collected on formvar-carbon-coated copper grids and stained with uranyl acetate and lead citrate by standard procedures. Observations were made on a Tecnai 10 TEM (FEI) and images were captured with a Veleta CCD camera and processed with SIS iTEM (Olympus).

Bacteria samples were collected by centrifugation from liquid cultures, fixed for 1 h at room temperature in 2.5% glutaraldehyde in culture medium, rinsed in 0,1 M cacodylate buffer, and postfixed in 2% OsO4 in the same buffer. After serial dehydration in increasing ethanol concentrations, samples were embedded in agar 100 (Agar Scientific Ltd., Stansted, UK) and left to polymerize for 2 days at 60 °C. Ultrathin sections (50–70 nm thick) were collected in Formvar–carbon-coated copper grids by using a Leica EM UC6 ultramicrotome and stained with uranyl acetate and lead citrate by standard procedures. Observations were made on a Tecnai 10 transmission electron microscope (FEI). Morphometric analyses and images processing were performed using the SIS iTEM (Olympus) software. Cell wall length were measure on all cells presented in the images, for each cell, 4 measures were taken at each cardinal point. This methodology led to extreme reading when phenomenon of failed cell division was encountered.

Whole bacterial cells were applied to glow discharged carbon‐coated Formvar copper grids. Bacterial cells were negatively stained with 4% ForMol. Observations were done on a Tecnai 10 (FEI) microscope coupled to a Veleta charge‐coupled device (CCD) camera (Olympus iTEM), and images were captured and analyzed using SIS Olympus iTEM software. Whole bacterial cells were applied to glow discharged carbon‐coated Formvar copper grids and negatively stained with 4% Uranyl acetate. Observations were done on a Tecnai 10 (FEI) transmission electron microscope coupled to a Veleta CCD camera (Olympus iTEM), and images were captured and analyzed using SIS Olympus iTEM software. For SEM, samples were fixed overnight at 4°C in glutaraldehyde 2.5%, 0.1 mol/L cacodylate buffer (pH 7.2), and postfixed in OsO₄ (2%) in the same buffer. After serial dehydration samples were dried at critical point and coated with platinum by standard procedures. Observations were made in a Tecnai FEG ESEM QUANTA 200 (FEI) and images were processed by SIS iTEM (Olympus) software.