As indicated above, the needs of the projects for cultured endothelial cells have decreased sharply since our previous submission, refiecting 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 isolafion procedures will importanfiy contribute to our interpretafion ofthe results. A. Isolation of mouse neutrophils. Currently, several different approaches are in use among the different projects. (i). For micropipette studies, a drop of blood is collected into low endotoxin buffer (0.1%BSA in HBSS, supplemented with lOmM HEPES, pH 7.4, 290-300mOsm);neutrophils are picked out visually and verified subsequenfiy using a Vybrant dye. These cells constitute our """"""""gold standard"""""""" for unactivated neutrophils, due to the minimal intervenfion involved in collecfion and the ability to direcfiy observe and quantify their behavior. (ii). For flow cytometry, mouse blood is collected from anesthetized (Nembutal, 65mg/kg/ ip) mice by heart puncture. Blood (2.5ml) is diluted hwice with buffer (lOmM HEPES in HBSS) and layered over 2.5ml NycoPrepI .077A. After spinning (600g, 20min), mononuclear cells form the band, and RBCs plus neutrophils form the pellet. These two components are separated, and RBCs lysed in both components using 1:6 dilution with PBS for 30sec followed by an appropriate volume of 4x PBS to restore osmolarity. They are then washed 3x with 0.1% BSA in HBSS supplemented with lOmM HEPES). To remove neutrophils we use magnefic extracfion (columns and reagents from Miltenyi Biotec, Auburn, CA). The pellet fracfion is resuspended in 90pl buffer together with 30pl FcR blocking reagent and 30pl biofinylated anfiCD49D (to remove eosinophils). Following 10 min incubation at 4C, 90pl buffer and 60pl anfi-biotin Microbeads are added;further lOmin incubation at 4C is followed by washing and resuspension in 500pl buffer. Cells are the separated magnetically (MACS separator). (Similar procedures are used to separate lymphocytes or monocytes from the band component, using appropriately chosen anfibodies). (iii). For cells to use in crawling assays, (a) From whole blood. Whole blood collected as above by cardiac puncture is diluted with an equal volume of 3% Dextran70 and allowed to sediment at room temperature for 30-60min. The top layer is then transferred to a Falcon tube and spun at 200g for lOmin at 25C. The supernatant is aspirated and the pellet resuspended with PBS, and Ficoll-paque added. After spinning at 400g for 30min at 25C, supernatant is aspirated, washed in equal volume of 0.2% PBS (30sec) then 1.6% PBS (30sec), then spun 200g, lOmin, 4C. This wash sequence is repeated a total of 3x. The supernatant is discarded and the neutrophil pellet resuspended into HBSS. (b) From bone marrow. Bone marrow is flushed from mouse tibia and/or femur with HBSS, supplemented with 20mM HEPES and 0.5% FCS. Bone marrow is disaggregated by repeated aspirafion thru an 18ga needle and pelleted at 400g for 5min. RBCs are lysed (0.2% NaCI and tonicity restored with 1.6% NaCI. Cell suspensions are clarified using a 70 pm cell strainer (BD, Inc), washed once in HBSS, and 5 pi is layered onto 5ml Percoll solution (Amersham). Cells are centrifuged lOOOg, 30min, and mature neutrophils are recovered from the bottom layer of the gradient Cells are rinsed 2x in HBSS then resuspended in HBSS supplemented with lOmM HEPES and ImM CaClj. We will compare outcomes of these procedures, with the initial goal of verifying that the activation state of cells isolated by these procedures is not significantly different. We will consult with each project to identify which metrics to use for definition of activation state (L-selectin surface expression density, cell morphology. We will also use flow cytometry to compare surface expression density of selected surface molecules (primarily (32 and 3i integrins) of the cells isolated by the different processes, as addifional validation that the cell populations being studied are comparable. In an iterative interacfion with the projects, the core technician will also work to modify protocols to improve yield of unactivated cells. Throughout this process, the core technician will consult and interact with personnel from the Rochester projects to disseminate information as it becomes available. She will also actively participate in our regular PPG meetings to present updated information to the group. Protocols and updates will be exchanged with Project 1 personnel by regular emails, and at the regular retreats where all project personnel are together. The PPG has an established history of travel between U Penn and Rochester to exchange technical information, and we will expect to do this if needed. B. HL60 cells. Currently, Projects 1-3, and 5 will use differentiated HL60 cells. These projects have experience with this cell line, but as described in the current submission we plan to characterize the mechanical as well as chemical properties of these cells, and to use siRNA and associated technology to modify their functional responses, we consider that it would be prudent to expand the above plans for standardizing mouse cell handling to HL60 cells. The techniques being used currently by Project 2 will be transferred to Core B, and, as above, the core technician will work with personnel from each project to maintain cells and standardize procedures for culture and differentiation. Heterogeneity within the dHL60 population is a concern that is currently being addressed by selecting to study those cells whose size is consistent with the neutrophil-like subpopulafion. HL60 cells are purchased from American Type Culture Collection and maintained in RPM11640 (Gibcolnvitrogen) supplemented with 1% penstrep and 10% heat-inactivated FBS, in 5% CO2 at 37C. They are diluted in new media when they reach 8-10 x 10^ cells/ml: doubling rate is 22-24 hrs. Differentiation is undertaken by (i) seeding cells at 1 x 10^ cells/ml in RPMI with 10% FBS, 1% penstrep and 1.3% DMSO. (ii) allowing cell growth for 4-7 days without changing the media. Cell density should plateau after 4 days at = 5-9 X 10^ cells/ml. (iii) The cells will differentiate into two populations;larger size (= 9pm diameter) is neutrophil-like and constitutes the principal population between days 5-6 of differentiation. Motility and transfection studies are being done on cells from days 5-6, based on Mac-1 expression and transfection efficiency with the currently used transfection techniques. Standardization of procedures for isolafion and use of human neutrophils will not at this time be transferred to the core. Procedures for isolation of non-activated human neutrophils were developed and carefully defined in earlier years of the PPG by Project 3 personnel in collaboration with the late Dr. Knauf. Those techniques have already been transferred to Projects 1, 2 and 5. Given the need to use these cells immediately upon isolafion, there is no substantive role for the core in this aspect of cell isolation and handling. C. Endothelial cells (HUVECs). Project 2 plans protocols using ECs, hence the Core will confinue to support this requirement. Fully characterised HUVECs are purchased from VecTechnologies Inc from a specified stock of pooled cells, ensuring uniformity of cell source for all protocols. They are routinely confirmed to be positive for Factor VIII expression and LDL incorporation. A stock of these cells is available in the core: cells will be transferred to Project 2 as needed. D. Gene-altered mouse populations. Project 3 will receive gifted knockout mice from outside investigators that will be used by Projects 3 and 1. Project 2, as described and budgeted there, is making and will use, a series of gene-altered animals. Importantly, these animals will also be used by all the other projects, at various times. Thus, without assigning some of the responsibility for these mice to a Core facility. Project 2 would be required to carry the needs (both in terms of planning breedings, and carrying costs) for all the other Projects'usage that is not explicitly collaborative with Project 2. Similariy, Project 3, would be responsible for making mice available to Project 1. Given that the personnel in Core B interact with Vivarium staff on a regular basis, and have the relevant experience in all aspects of mouse husbandry, this appears to be the logical place to most efficiently put this responsibility. We have included in the budget for years 3-5 expenses to support production of enough mice to support the needs of the other projects with either Project 2's integrin knock-in /FRET mice, and the Rapl and CalDAG-GEFI knockouts being acquired by Project 3.
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