Quantifying Endothelial Cell Adhesion Markers

At the basal state and after stimulations, non-permeabilized cells were detached by 5 mM EDTA, washed, and re-suspended in 0.5% BSA solution. The cells were treated and incubated with anti-VCAM-1 PE conjugate (Santa Cruz Biotechnology, sc-13160, 1:100) and with anti-ICAM-1 FITC conjugate (Santa Cruz Biotechnology, sc-107, 1:100) as previously described (Ucci et al., 2019 (link)). One test tube for the basal state and one for the TNF-α-treated condition were incubated with anti-VCAM-PE and anti-ICAM-FITC isotypes (normal mouse IgG2a FITC-conjugated and normal mouse IgG1 PE-conjugated, Santa Cruz Biotechnology, 1:100) as negative controls. Flow cytometry analysis was performed on a BD FACS Canto II flow cytometer (BD Bioscences) and for each sample 1 × 10⁴ events were analyzed using FACSDiva v 6.1.3 (BD Biosciences) and FlowJo 8.3.3 software (Tree Star Inc., Ashland, United States). All the results are expressed as mean fluorescence intensity (MFI) ratio ±standard deviation (SD). Each value was calculated by dividing the MFI of positive events by the MFI of negative events (MFI of secondary antibody). The experiment was carried out on four different strains for C-HUVECs and four different strains for GD-HUVECs, each in technical triplicate.