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Copyright Thomas J. Lucier 2005. To customize this document, download it to your hard drive from Thomas J. Lucier s web site at www.thomaslucier.com/optionforms.html. The document can then be opened, edited, and printed using Microsoft Word or another popular word processing application.

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Note: The antibody that is utilized in this protocol has already been conjugated to a uorescent marker. However, it is possible that under different circumstances, the cell surface marker would be bound by an antibody that would then be attached to a secondary antibody containing the uorescent signal. Protocol variations for this have been noted below. Reagents and Materials Sterile EBs, 13- to 15-day culture(with medium changed every 2nd 3rd day) Trypsin, 0.1% in PBSA TNS: trypsin neutralizing solution FBS, 5% in PBSA Conical centrifuge tubes, 15 ml Take EBs, which have been cultured on nonadherent dishes for 13 15 days, from the CO2 incubator and place in the hood. (b) Remove the suspension of cells in medium and place in 15-ml conical centrifuge tubes. (c) Allow the EBs to settle out of the medium (5 10 min). (d) Aspirate the medium. Try to maximize the amount removed without disturbing the pellet. (e) Add 7 ml trypsin to 3 to 4 15-ml tubes. Repeat as necessary for additional tubes. Cap tubes very tightly and put on an xyz shaker in an CO2 incubator for 5 min. (f) Remove tubes from the incubator and pipette up and down strongly to dissociate EBs. If necessary, place back in the incubator for an additional 2 min. (g) Add 7 ml TNS to each tube. (h) If necessary, pour the cells through a cell lter to remove any clumps.

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Ever-decreasing cost to deploy should drop to about $1400 per customer averaged over the years 2004 2008. Extending FTTH to 50% of these three carriers residential customers and 80% of their business customers in suburban areas of the top 20 U.S. states (costing the carriers $45 to $50 billion) would improve their revenues by $9.5 billion by the end of the deployment, which would take 5 to 7 years. Annual savings in operating expenses would reach $5.5 billion by 2008 and $7.8 billion by 2010. These OPEX savings would be gained by such things as a 100% expense reduction at the central of ce (CO), and 30 to 70% reduction in customer service and network maintenance expenses.

abstractions. Although it is often technically feasible to re-inject used groundwaters back into the aquifers from whence they were originally abstracted (see Section 7.4.2), it is often much cheaper to discharge such waters to the nearest surface watercourse. By contrast, it is extremely rare for spent surface waters to be injected into an aquifer. The relative virtue of a mode of water use which results in a high-percentage return ow must be judged from an holistic environmental perspective, rather than being regarded as inherently virtuous without further scrutiny.

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