For baseline secretion, static assays (image analysis in Figure 3 and Western blotting in Figure 4) show a clear defect in SNAP23 Het mice, whereas the kinetic assay of released mucin shows no difference between WT and Het mice.
Because adhesion in the dynamic in-vitro model described here is TNF- and chemokine-dependent, it may be a good alternative or supplement to static assays for leukocyte-endothelial cell interactions.
For static cytoadhesion assays Chinese hamster ovary cells defective in glycosaminoglycan biosynthesis (CHO-745, American Type Culture Collection) were transfected using Lipofectamine 2000 (Invitrogen) complexed with pAcGFP1-N1 (BD Biosciences Clontech), pAcGFP1-N1-cd36, pAcGFP1-N1-icam-1, pAcGFP1-N1-p-selectin or pEGFP-vcam1.
For static adhesion assays, a luciferase reporter plasmid was used to detect transfected cells: cells were allowed to adhere to immobilized fibronectin or recombinant human intracellular adhesion molecule, non-adherant cells washed off, and adherant cells quantitated by luciferase assays.
Methodology includes static assays in which membrane's samples were immersed in a cleaning solution for over 12 or 24h at a temperature of 25 to 40°C.
Static assays were performed to assess the response to other secretagogues.
We have analysed the role of polymorphic variants of ICAM-1 in severe falciparum malaria using static and flow based adhesion assays and CD36 in static assays only.
For stimulated secretion, a static assay of intracellular mucin content (image analysis in Figure 3B) shows impaired release in SNAP23 Het mice that is roughly commensurate with the level of SNAP23 protein.
Microfluidic-based assays should, therefore, be better suited than the classical static assay systems for studying endothelial cells and their interactions with blood cells, cancer cells and circulating stem and progenitor cells.
For example, CD36, thrombospondin (TSP), chondroitin sulfate A (CSA) and intercellular adhesion molecule-1 (ICAM-1) are widely expressed on the surface of endothelium in various organs and found to be able to mediate stable bindings in static assay.
