Cell quest software version 4

IEC-6 cells were plated into 96-well plates (2.0 × 10³ cells/well) and treated with almond ASE and CD-ASE for 24 h in LPS + IFN-stimulated IEC-6 cells. After the treatment with the tested products, IEC-6 cells were collected and washed with phosphate buffered saline (PBS), and then fixing solution was added to cells for 20 min. Cells were subsequently incubated in a fix perm solution for 30 min, and anti-COX-2 (BD Transduction Laboratories, Milan, Italy) and anti-iNOS (BD Transduction Laboratories, Milan, Italy) antibodies were then throw in for 1 h. Then, the secondary antibody, in fixing solution, was added to IEC-6 cells and a fluorescence-activated cell sorter (FACSscan; Becton Dickinson, Milan, Italy) and analyzed by Cell Quest software (version 4; Becton Dickinson, Milan, Italy) to evaluate the cell fluorescence [25 (link)].

IEC-6 cells were plated into 96-well plates (2 × 10³ cells/well) and treated with the peptides in inflammatory conditions, as previously described, for 24 h in order to evaluate COX-2, iNOS, HO-1, and SOD expression and nitrotyrosine formation. For this analysis, the cells were collected and washed with phosphate-buffered saline (PBS). Fixing solution was added to cells for 20 min and then incubated in fix/perm solution for a further 30 min. Anti-COX-2 (BD Transduction Laboratories, Milan, Italy), anti-iNOS (BD Transduction Laboratories, Milan, Italy), anti-HO-1 (Santa Cruz Biotechnologies, Dallas, TX, USA), anti-SOD (Santa Cruz Biotechnologies, Dallas, TX, USA), and anti-nitrotyrosine (Merck Millipore, Milan, Italy) antibodies were then added for 1 h. The secondary antibody, in fixing solution, was added to the cells and cell fluorescence was then evaluated by a fluorescence-activated cell sorter (FACSscan; Becton Dickinson, Milan, Italy) and analyzed by Cell Quest software (version 4; Becton Dickinson, Milan, Italy) [35 (link)].

The probe 2′,7′-dichlorofluorescein-diacetate (H₂DCF-DA) was used to evaluate ROS intracellular production [26 (link)]. IEC-6 cells were plated in 24-well plates (8.0 × 10⁴ cells/well). After adhesion, almond ASE and CD-ASE (50–5 μg/mL) were added for 1 h alone and then simultaneously to LPS (10 μg/mL) plus IFN (10 U/mL) for 24 h. In other experiments, the cells were treated with the tested products, at the same concentrations, alone for 1 h and then simultaneously to H₂O₂ (1 mM) for another 1 h. IEC-6 cells were then collected, washed with PBS and incubated in PBS plus H₂DCF-DA (10 μM). After an incubation of 15 min at 37 °C, cell fluorescence was evaluated using a fluorescence-activated cell sorter (FACSscan; Becton Dickinson, Franklin Lakes, NJ, USA), and was analyzed by Cell Quest software version 4 (Becton Dickinson, Milan, Italy) [27 (link)].

ROS levels were evaluated by means of the probe 2′,7′-dichlorofluorescin-diacetate (H₂DCF-DA) [31 (link)]. HaCaT cells were plated in 24-well plates (8.0 × 10³ cells/well) and, after adhesion, were treated as previously described, for 24 h. The cells were then collected, washed with phosphate buffered saline (PBS), and then incubated in PBS containing H₂DCF-DA (10 μM). After 15 min at 37 °C, cell fluorescence was evaluated using a fluorescence-activated cell sorter (FACSscan; Becton Dickinson, Franklin Lakes, NJ, USA) and analyzed with Cell Quest software (version 4; Becton Dickinson, Milan, Italy).

After the cellular treatment, HaCaT were collected, washed with PBS, then incubated in fixing solution for 20 min and then in Fix Perm solution for 30 min. Anti-heme oxygenase-1 (HO-1; Santa Cruz Biotechnologies, Dallas, TX, USA), anti-NAD(P)H Quinone Dehydrogenase 1 (NQO-1; Santa Cruz Biotechnologies), anti-Bax (Santa Cruz Biotechnologies, Dallas, TX, USA), anti-Bcl-2 (Santa Cruz Biotechnologies, Dallas, TX, USA) or anti-nitrotyrosine (Merck Millipore, Milan, Italy) antibodies were then added for 1 h. The secondary antibody was added to HaCaT cells in fixing solution and cell fluorescence was then evaluated by a fluorescence-activated cell sorter (FACSscan; Becton Dickinson, Milan, Italy) and then elaborated by Cell Quest software (version 4; Becton Dickinson, Milan, Italy) [32 (link)].

Antibodies used for both surface and intracellular staining were diluted at 1:250. The following mouse mAbs were used for flow cytometric analysis: CD4-FITC (cat. no. 553047), CD11c-FITC (cat. no. 553801), CD40-PE (cat. no. 553791), CD80-PE (cat. no. 553769), I-A^b-PE (cat. no. 553552) (BD Biosciences, San Diego, CA, USA), IL-17A-APC (cat. no. 17-7177; eBioscience, Inc., San Diego, CA, USA). To detect intracellular IL-17A, the cells were re-stimulated for 6 h with 50 ng/ml PMA, 1 μg/ml ionomycin, and 1 μl/ml Golgiplug at 37°C and 5% CO₂. Subsequently, the cells were intracellularly stained after permeabilization of the cells using Cytofix/Cytoperm kits (BD Biosciences, San Diego, CA, USA). The stained cells were observed using a flow cytometer (FACSCalibur or BD Accuri C6 Plus; BD Biosciences, San Diego, CA, USA) gated on live CD11c⁺ or CD4⁺ cells. The data were analyzed using CellQuest software version 4.0.2 (BD Biosciences, San Diego, CA, USA).