Fitc anti human icam 1 cd54

1 × 10⁵ epithelial target cells were washed and re-suspended in the cell staining buffer (Biolegend) containing different dye-conjugated primary antibodies at 1 μg/ml or 5 μg/ml for 30 min at 4°C. Cells were then washed and stained with the Zombie NIR (cell death marker, Biolegend). The stained cells were analyzed by flow cytometry with a FACSCanto II cytometer (BD Biosciences). Primary antibodies targeting the various surface ligands and receptors as well as the corresponding control include: FITC anti-human HLA-A,B,C (Clone W6/32, Biolegend), PE anti-human HLA-E (Clone 3D12, Biolegend), FITC anti-human ICAM-1/CD54 (Clone DX2, Biolegend), FITC anti-human Fas/CD95 (Clone DX2, Biolegend), APC anti-human Nectin-2/CD112 (Clone TX31, Biolegend), APC anti-human PVR/CD155 (Clone SKII.4, Biolegend), PE anti-human MICA (Clone 159227, R&D Systems), PE anti-human MICB (Clone 236511, R&D Systems), PE anti-human ULBP-2/5/6 (Clone 165903, R&D Systems), FITC Mouse IgG1 κ Isotype Control (Clone MOPC-21, Biolegend), APC Mouse IgG1 κ Isotype Control (Clone MOPC-21, Biolegend), and PE Mouse IgG2b κ Isotype Control (Clone MPC-11, Biolegend). Cell death analysis by Annexin V staining was performed using the FITC Annexin V detection kit (Biolegned) following the manufacturer’s protocol.

To determine the protein expression of adhesion molecules on the HUVEC membrane, cells were washed with PBS, detached using trypsin, washed with PBS, and re-suspended in ice-cold FACS buffer (PBS supplemented with 5% FCS). The cells were divided equally into separate FACS tubes. The cells were stained using the following antibodies: PE-conjugated anti-human E-selectin (CD62E) (Biolegend), APC-anti human VCAM-1 (CD106) (Biolegend), FITC- anti human ICAM-1 (CD54) (Biolegend), and IgG isotope controls (IgG isotope controls, Biolegend) for 30 min on ice. The cells were washed once and resuspended in FACS buffer. Samples were analyzed using a MACSQuant Analyzer 10 system (Miltenyi Biotech, San Diego, CA, USA). Multi-color compensation was calibrated using positive control cell population (LPS activated HUVECs). Data were presented as the Geometric Mean of Fluorescence Intensity (MFI).