Glutamine

HUVECs were cultured in 12 well plates (Thermofischer Scientific, USA) at a density of 4 × 10⁴ cells/well and incubated in Endothelial Cell Growth Medium with antibiotics, glutamine, and heparin (PELO Biotech) supplemented with 2.5% FBS. 17-19 h later, the cells were washed and incubated in medium with 4 mM H₂O₂ (Merck, Darmstadt, Germany), 200 nM staurosporine (Abcam), 2 µg/mL HrA2 and vehicle for 2 and 24h. The morphology of HUVECs was observed under an inverted phase contrast microscope (microscope: AXIO Vert.A1, Zeiss, camera: AxioCam ICc1).

The expression of ICAM (CD54) and VCAM (CD106) was analyzed by flow cytometry using Flow Cytometer BD FACS Canto II (Beckton Dickinson). For this, HUVEC were maintained as mentioned above and were seeded in a 12-well plate at a density of 7 × 10⁴ in Endothelial Cell Growth Medium with antibiotics, glutamine, and heparin (PELO Biotech, Martinsried, Germany) supplemented with 2.5% FBS. 17–19 h later, the cells were washed and treated with 10 ng/mL TNFα (RnD Systems), 2 µg/mL HrA2 and vehicle for 4 h, 8 h and 24 h. After treatment, HUVECs were washed with phosphate buffered saline (PBS) (Thermofischer, Scientific, Waltham, MA, USA), detached with trypsin 0.05% EDTA (Thermofischer, Scientific) and resuspended in 48 µL PBS plus 0.1% bovine serum albumin (BSA) (Merck, Kennelworth, NJ, USA), stained with 1 µL Pacific Blue conjugated antibody CD54 and 1 µL APC conjugated antibody CD106 per sample. After 30 min of incubation at 4 °C, in the dark, the cells were centrifuged at 500 g for 5 min and resuspended in 300 µL PBS plus 0.1% BSA.