Pe mouse anti human cd54

The HMEC-1 were seeded onto 96-well plates at a concentration of 3·10⁴ cells per well, left to grow for 24 h to the 100% confluence, and then incubated with LPS in various concentrations (0.01, 0.1, 1.0, 10, 100 µg·mL⁻¹). After 24 h, the cells were stained with mouse anti-human CD54-PE (BD Pharmingen, San Jose, CA, USA), according to the manufacturer’s protocol, and with Hoechst 33,342 (Life Technologies, Warszawa, Poland) for 10 min. The expression of surface ICAM-1 was observed by fluorescence microscopy (a ScanR screening system) in randomly selected 8 visual fields for each well and was analyzed using Columbus 2.4.2 Software (Perkin Elmer, Waltham, MA, USA). The experiments were performed in triplicate and repeated three times.

The HMEC-1 were seeded onto 96-well plates at a concentration of 3·10⁴ cells per well, in 200 µL of medium per well and left for 24 h to grow to the 100% confluence, and then were treated as follows: AXT (10 µM, 3 h), TNF-α (10 ng mL⁻¹, 24 h), AXT with TNF-α pretreatment and AXT followed by TNF-α. Cells in fresh MCDB131 medium and DMSO were taken as a negative controls. Then fixed HMEC-1 (2.5% glutaraldehyde, 4 min) were incubated in the dark with mouse anti-human CD54-PE (BD Pharmingen) and stained with Hoechst 33342 (Life Technologies) for 30 min. The overexpression of surface ICAM-1 molecule and cell nuclei were observed by fluorescence microscopy (a ScanR screening system) in randomly selected six visual fields for each well. The images were analysed using Columbus 2.4.2 Software (Perkin Elmer). The results of ICAM-1 expression were presented as mean fluorescence intensity per cell for each analysed group.

Flow cytometric analysis was preformed using a FACS Canto II flow cytometer (BD Biosciences, Franklin Lakes, NJ, United States). For staining of cell surface expression of ICAM-1, unspecific binding was blocked with 5% FCS in PBS for 5 min at room temperature (RT). Cells were stained with fluorophore-conjugated antibodies directed against ICAM-1/CD54 (1:25 PE mouse anti human CD54, BD Pharmingen). Data were analysed using FlowJo software, version 10 (FlowJo™, BD).