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Briefly, 4 × 104 B16F10 cells per milliliter were added to 90-mm tissue culture plates and allowed to grow up to 90% confluency.
Erythrocytes were resuspended in PBS or experimental plasma at physiologic concentrations (5 × 10 cells/mL) and 2 × 10 Ad5 particles per milliliter were added.
Ten units of enzyme per milliliter were added to a solution of 10 mM NAD+ or APAD+ in 0.1 M Tris-HCl (pH 8) containing either 100 mM d-glucose- d1 or 6% (v/v) 2-propanol d8 and the mixture incubated at room temperature until the absorbance at 340 nm stopped increasing (approximately 30 min).
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Five milliliters were added to flasks for culture, another 3 ml were collected in an ethylenediamine tetraacetic acid-coated tube (Becton Dickinson, Cockeysville, MD, USA) for immunophenotyping, and the remaining blood was added in a sterile tube.
Half-milliliter volumes were added to a 10 mm Hellma GmbH&Co quartz cuvette, and fluorescence was measured with a Perkin-Elmer LS5 Luminescence Spectrometer.
Two milliliter of ACN were added for protein precipitation.
Four milliliter of toluene were added to the solution and vortexed for 15 20 s.
Ten micrograms per milliliter of BrdU were added to the culture medium for 24 h.
Two milliliters of inoculum were added to each root system.
Three milliliters of iodomethane were added, and the mixture was allowed to react for 48 h at 60 °C, under nitrogen gas flow and stirring.
Two microgram per milliliter of BD GolgiStop were added at 2 h before cell harvesting.
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