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.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Program Projects (P01)
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Heart, Lung, and Blood Initial Review Group (HLBP)
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University of Rochester
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Vats, Kanika; Marsh, Graham; Harding, Kristen et al. (2017) Nanoscale physicochemical properties of chain- and step-growth polymerized PEG hydrogels affect cell-material interactions. J Biomed Mater Res A 105:1112-1122
Henry, Steven J; Crocker, John C; Hammer, Daniel A (2016) Motile Human Neutrophils Sense Ligand Density Over Their Entire Contact Area. Ann Biomed Eng 44:886-94
Svetina, Saša; Kokot, Gašper; Kebe, Tjaša Švelc et al. (2016) A novel strain energy relationship for red blood cell membrane skeleton based on spectrin stiffness and its application to micropipette deformation. Biomech Model Mechanobiol 15:745-58
Marsh, Graham; Waugh, Richard E (2016) A simple approach for bioactive surface calibration using evanescent waves. J Microsc 262:245-51
Rocheleau, Anne D; Wang, Weiwei; King, Michael R (2016) Effect of Pseudopod Extensions on Neutrophil Hemodynamic Transport Near a Wall. Cell Mol Bioeng 9:85-95
Rocheleau, Anne D; Cao, Thong M; Takitani, Tait et al. (2016) Comparison of human and mouse E-selectin binding to Sialyl-Lewis(x). BMC Struct Biol 16:10
MacKay, Joanna L; Hammer, Daniel A (2016) Stiff substrates enhance monocytic cell capture through E-selectin but not P-selectin. Integr Biol (Camb) 8:62-72
Hind, Laurel E; Lurier, Emily B; Dembo, Micah et al. (2016) Effect of M1-M2 Polarization on the Motility and Traction Stresses of Primary Human Macrophages. Cell Mol Bioeng 9:455-465
Hughes, Andrew D; Marsh, Graham; Waugh, Richard E et al. (2015) Halloysite Nanotube Coatings Suppress Leukocyte Spreading. Langmuir 31:13553-60
Lim, Kihong; Hyun, Young-Min; Lambert-Emo, Kris et al. (2015) Visualization of integrin Mac-1 in vivo. J Immunol Methods 426:120-7

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