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Reagents and Materials Sterile RPMI 1640 medium supplemented with 5% FBS Centrifuge tubes, 50 ml (Falcon) Polystyrene tubes, 5 ml (Falcon) Antibodies: CD14-FITC, CD31-PE, CD34-APC, CD44-FITC, CD45-APC, CD71APC, mouse anti-human CD105, goat anti-mouse IgG-FITC Nonsterile Formalin 2% in PBSA (prepare fresh, keep at 4 C) Incubator at 37 C, 5% CO2 Hemocytometer Vortex mixer Protocol (a) Prepare cells according to Protocol 13.5 until step (m). (b) Wash cells twice in RPMI-FBS medium. (c) Count an aliquot of the cell suspension using a hemocytometer and determine cell viability through dye exclusion with Trypan Blue. Count unstained cells. (d) Centrifuge cells at 300 g for 10 min at 4 C, aspirate supernate, and resuspend cells in RPMI-FBS at a concentration of 1 107 cells/ml in a 50-ml Falcon tube. (e) Add 50 l of this cell suspension (500,000 cells) in each 5-ml Falcon tube. (f) Add 2 l of each desired CD stock solution (CD45-APC, CD44-FITC, CD31PE, CD34-APC, CD14-FITC, CD71-APC, CD105; concentration of each: 0.5 g/ l) into each corresponding 5-ml Falcon tube. (g) Incubate for 30 min on ice in the dark. (h) Centrifuge at 300 g for 5 min at 4 C and aspirate supernate. (i) Wash in 1 ml RPMI-FBS medium. (j) Centrifuge at 300 g for 5 min at 4 C. (k) Aspirate medium. (l) Add 1 ml RPMI-FBS medium and resuspend cells gently. (m) Centrifuge at 300 g for 5 min at 4 C. (n) Carefully aspirate supernate. (o) Resuspend cells in 1 ml RPMI-FBS medium; place on ice, except for tubes designated CD105 (go to Step (p) for non-labelled antibodies; here for CD105; and for others continue at (u)). (p) All tubes designated CD105: add 50 l RPMI-FBS and 2 l secondary antibody (polyclonal goat anti-mouse IgG-FITC); incubate on ice for 30 min in the dark.
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Hiding selection highlighting You can hide selection highlights in order to edit objects without viewing their highlighting. Hiding highlights enables you to see how artwork will appear in its final state while you are selecting and editing objects.
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White space. Uncluttered space on the page. Margins around text and images is white space.
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IPv6 will not be cut over in an hour s time. A period of transition will take place. In
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18.3.2.4 Global Synchronization. GS is the implementation of the synchronous protocol S-MAC proposed in [6]. It tries to synchronize all devices in the networks by transmitting a SYNC frame. A SYNC is a command frame broadcasted with a schedule information like a beacon. The difference is that the SYNC is not transmitted at every wakeup interval and CSMA/CA is used when it is transmitted. Therefore, it is free from the continuous collision problem. However, a longer time than the time for a beacon should be reserved to receive the SYNC. Figure 18.7 presents an example of time lines of GS. The time line of GS is divided into wakeup intervals. Each wakeup interval is started from a short active duration and followed by a long inactive duration. The active duration is divided into two time spaces. The rst time space is for a SYNC, and the second time space is for a request to send (RTS). When a device transmits a SYNC in the active duration, other devices adjust their clocks.
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virtually the same as before. Based on this fact, we propose to use a maxTSC that is much larger than otherwise needed. The motivation is that: 1. A predetermined maxTSC precludes the need for exchanging local TSC information to get the maxTSC after the rst round of time slot assignments. As such, both overhead and delay can be reduced. When maxTSC is predetermined, a node does not need to select the smallest possible time slot in the rst round of time slot assignment. It can select any available slot. This facilitates the optimization of time slot assignment (discussed later). 2. A large enough maxTSC guarantees there are enough time slots to be assigned even for a very irregular network topology (e.g., the node density is very high in some area). Yet this large maxTSC will not introduce additional delay as can be seen from the example of tripling the maxTSC. 3. A small maxTSC leads to unfairness in time slot assignment. This can be seen from Fig. 15.3. To understand this, let us compare the different results of 7 slots allocated to 6 nodes and 70 slots allocated to 6 nodes. Apparently, we can do much better in the second case in terms of fairness. 4. With a large maxTSC, dynamic activities such as node joining, node leaving/failure, mobility, and sleeping mode can be better handled. All those activities require the adjustment of time slot assignment. A large maxTSC not only makes such adjustment possible but also easier and smoother.
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2.3 The First Optical-Based Calculation Techniques 33
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