As indicated above, the needs of the projects for cultured endothelial cells have decreased sharply since our previous submission, reflecting the refining and further focus of aims in all the projects in response both to new data and to issues raised in the reviews. In fact, only Project 2 of the Rochester projects plans aims requiring HUVECs. This requirement will continue to be supported by the core (amplified below). Concomitantly, there has been a significant expansion in plans to use isolated mouse leukocytes, both from WT animals and from the various gene-altered models. Uses range from micropipette studies on individual leukocytes isolated from a drop of mouse blood (Project 3), to isolation and identification of labeled populations by flow cytometry and/or by microscopic inspection (Projects 2, 3 and 4), and isolation of larger numbers of cells for studies of cell motility on defined surfaces/molecules (Projects 1, 2, 3). An emerging concern is that these requirements have to date been met by different methods of isolation of the relevant leukocyte population. Inasmuch as the goals of this Program Project are dependent on the expectation that data from the various projects are interchangeable, it has become clear that we should pro-actively compare and standardize these various methods, so as to provide uniform procedures that can be used by all projects. Our major goal is to standardize all approaches so as to provide cells in the same (unactivated) state, using appropriately selected measures (below). Where it remains appropriate to use different cell isolation approaches (e.g., one drop of blood, which can be collected non-invasively from a knock-in animal, provides excess cells for pipette studies in Project 3, and retains the mouse for future work), then having identified any differences in relevant outcomes due to the isolation procedures will importantly contribute to our interpretation ofthe results.
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