Adhesion of PBMC on HUVEC 6 h after damage compared to untreated and TNF-α stimulated HUVEC.

A-F: representative images of adhered PBMC on HUVEC. Left images (A, C, E) adhered PBMC on non-affected HUVEC. Right images (B, D, F): adhered PBMC on affected HUVEC. A, B: unheated stamp control (mechanical damage), C, D: thermal damage, E: untreated HUVEC as control, F: TNF-α-treated (10 ng/ml) HUVEC. Small rounded PBMC that were counted are indicated with white arrows. Larger round cells that cannot be identified clearly as either larger PBMC or dead round HUVEC, were not counted for this analysis and are indicated by black arrows in D and F. G: quantification of adhered PBMC cells per cm². Na = non affected, a = affected, (-) = untreated HUVEC, (+) = treated HUVEC with TNF-α, RT = mechanical damage at room temperature, 100°C = thermally affected (100°C) HUVEC using the two-ring stamp. The analysis showed a significant increase after treatment of HUVEC with TNF-α as well as after thermal damage compared to the corresponding negative control and compared to the number of adhered PBMC on mechanically damaged cells. As another negative control, the adhesion of PBMC on collagen-coated cover slips was used to illustrate that PBMC do not simply adhere on thermally compromised collagen. H: representative image of adhered PBMC on collagen after mechanical damage, I: adhered PBMC on thermal damaged collagen, J: quantification of adhered PBMC cells per cm² on collagen. There is no increase of adhered PBMC after affecting simply the collagen-coated coverslips. The experiments were performed in triplicates, but for the adhesion experiments on collagen which were performed in duplicates. PBMC on HUVEC were counted in n > 27 images, and n = 21 images were counted for adhesion on collagen. Scale bars: 200 µm.