Pe icam 1

Following 15d-PGJ₂ or MG132 treatments (18 h) and activation with TNF- α (0.2 ng/ml for 6 h), the EC were harvested to be stained for flow cytometric analysis. To block non-specific binding, EC were incubated in 2% BSA in PBS (blocking buffer) for 15 min before adding the antibodies. FITC-VCAM-1, PE-ICAM-1, and APC-E-selectin antibodies (BD, Rankling Lakes, NJ, USA) were used to label EC in 2% BSA in PBS. The antibodies were incubated for 30 min at room temperature. Following two washes with blocking buffer, the cells were fixed in 2% paraformaldehyde. Forward and side scatter gates were established to exclude non-viable cells and cell debris from the analysis. The mean fluorescence intensity of 2 × 10⁵ cells was analyzed in each sample. Auto-fluorescence signals generated by unlabeled cells were used as negative controls in each experiment. Flow cytometric analysis was performed on an Accuri C6 instrument and analyzed with CFlow^® Software (Accuri, Ann Arbor, MI, USA). The data were expressed as median fluorescence intensity of three independent experiments.

Following treatments with casein-derived hydrolysate (18 h) and activation with TNF-α (0.5 ng/ml for 6 h), the EC were harvested to be stained for flow cytometric analysis. To block non-specific binding, EC were incubated in 2% BSA in PBS for 15 min before adding the antibodies. FITC-VCAM-1, PE-ICAM-1 (BD, Rankling Lakes, NJ) and APC-E-sel (Bio Legend, San Diega, CA) antibodies were used to label EC in 2% BSA in PBS. The antibodies were incubated for 30 min at RT. Following washes, the cells were fixed in 2% paraformaldehyde. Forward and side scatter gates were established to exclude nonviable cells and cell debris from the analysis. The mean fluorescence intensity of 2 × 10⁴ cells was analysed in each sample. Auto-fluorescence signals generated by unlabelled cells were used as negative controls in each experiment. Flow cytometric analysis was performed on an Accuri C6 instrument and analyzed with CFlow® Software (Accuri, Ann Arbor, MI). The protein expression levels were expressed as median fluorescence intensity of three independent experiments.