Cytomics fc 500 instrument

Bacterial cell membrane integrity assessment was evaluated by measuring propidium iodide (PI) (Sigma, Milan, Italy) uptake using flow cytometry [47 (link)]. Briefly, bacterial cultures in the mid-log phase were diluted in MHB to 1 × 10⁶ CFU/mL, and were incubated at 37 °C with MIC and sub-MIC concentrations of the peptide for different time points. PI was added to all samples at a final concentration of 10 μg/mL. At the end of each time point incubation, the bacterial cells were analyzed using a Cytomics FC 500 instrument (Beckman-Coulter, Inc., Fullerton, CA, USA). Data analysis was performed using FCS Express3 software (De Novo Software, Los Angeles, CA, USA). Data are expressed as mean ± SEM.

Human DCs were generated from monocytes isolated from the PBMCs of healthy HLA-A2 donors using Histopaque^®-1077 (Sigma-Aldrich). The PBMCs were resuspended in x-Vivo medium (Lonza, Walkersville, MD, USA) containing 1% human serum (Sigma-Aldrich) at a cell density of 1×10⁷ cells/ml and plated in T75 flasks. The flasks were incubated in 5% CO₂ at 37°C for 90 min, and nonadherent cells were then gently resuspended and removed. The adherent cells were then cultured in LGM-3 medium (Lonza, Walkersville, MD, USA) containing 1% human serum, recombinant human GM-CSF (1,000 units/ml, R&D Systems, USA) and IL-4 (1,000 units/ml, R&D Systems, USA) for 5 days. Next, the adherent cells were harvested, counted, and resuspended in culture medium. The phenotypes of iDCs were determined by flow cytometry using PE-conjugated anti-CD83 (1:20; IM2218U; Beckman Coulter, Marseille, France), anti-HLA-ABC (1:20; IM1838U, Beckman Coulter) and PE-conjugated anti-HLA-DR (1:20; IM1639, Beckman Coulter) antibodies. The expression of cell surface markers was then determined by flow cytometry using the Cytomics FC 500 instrument (Beckman Coulter, Fullerton, CA, USA).