Culture, 6. Also measurement

FRET efficiencies and acceptor to donor ratios of FRET standards. The FRET efficiencies of six genetic constructs expressed in cell culture were evaluated by three different methods sRET, FLIM-FRET, and E-FRET. The acceptor to donor ratio (V/C) was also measured for each construct by the sRET method. Key C5Y, Cerulean-5 amino acid linker-Venus C17Y, Cerulean-17 amino acid linker-Venus C32V, Cerulean-32 amino acid linker-Venus CYC, Venus flanked on each side by a Cerulean VCV, Cerulean... 

Very little of 7T H7-galactose precursor was converted to a glucocerbroside or to a lactosylceramide under these culture conditions (21). The activities of the sulfotransferase and galactosyltransferase, enzymes responsible for the synthesis of the myelin-associated sulfo- and glycolipids, were also measured at different days in culture (fig. 4). The activities of these enzymes in homogenates of cells derived from the 15 day embryo were relatively low at 8 days but increased until reaching... 

Freshly excised buccal or sublingual tissues have also been used to generate dissociated cells. Hedberg et al. [49] used one such culture to measure the expression of alcohol dehydrogenase-3 in cultured cells from human oral mucosal tissue. Human buccal tissue was incubated with 0.17% trypsin in phosphate-buffered saline (PBS) at 4°C for 18 to 24 h to obtain dissociated primary keratinocytes, and subsequently these keratinocytes were seeded onto fibronectin and collagen-coated dishes in serum-free epithelial medium. 

At the end of growth in the Ni-containing medium, the cells were separated by filtration (0.4 jl Nuclepore) and dry weights were determined. An aliquot of the filtrate (<0.4 jl) was refiltered (<0.01) and the Ni in this fraction was taken as soluble. Measurements on each culture also included initial and final pH, cell density, and the initial and final Ni concentration in the filtered (<0.4 and <0.01 jl) exocellular medium. 

When investigating cell kinetics, it is very important to control precisely the physicochemical parameters of the medium, such as temperature, pH, dissolved oxygen and osmotic pressure. It is also important to define precisely the state of the inoculum, which may have a significant effect on the progress of the culture. During the culture, one measures at regular time intervals the concentrations of living and dead cells, of the major nutrients and metabolites and of excreted proteins. 

These investigations show clearly that extracellular production observed was not just a portion of the cell content and therefore not attributable to cell leakage or lysis. Myklestad et al. [26] also measured release rates of carbohydrate and amino acids from C. affinis and concluded that extracellular production is taking place in all growth phases in culture (Table 3). It should be noted that absolute release per cell was as high or higher in the exponential phase than in the stationary phase. 

To control whether stimulation was successful we continuously recorded the impedance spectra of the cell cultures for 1 h. As published before the stimulation causes an impedance increase at around 17.5 kHz [3]. Nevertheless, these measurements reflect an unrealistic state where the cells are cultivated on the electrodes. Therefore, we also measured the electrical properties of the medium without growing cells on the electrode to describe the status when the neutrophils arrive and exclude their chromatin in the wound. To determine the electrical medium properties we used the cultivation medium as a reference in a new well. The top layer of cell medium in the cell culture was discarded after a stimulation period of 1 h. The remaining bottom layer containing mostly NETs was suspended in the new well. For the unstimulated control sample this medium contains mostly unstimulated cells. All displayed spectra are representatives for one well. 

Next we applied this assay to the assessment of a nickel-titanium alloy [18]. We selected MOLT-3 that was the most sensitive against nickel ion (see Fig. 11). Nickel-titanium (NiTi) alloys were made by an arc melting method. Those alloys varied greatly in composition (Table 3) and phase (Fig. 12). First these alloys were sterilized by soaking them in 70 % ethanol solution. Then they were immersed in phosphate buffer and incubated at 36.5 °C for 25 h. We collected the extract and added it to a MOLT-3 culture plate (the ratio of extract to supernatant was 1 1). After a 4-day culture, we measured the viable cell number of each culture condition. The order of the viable cell numbers was coupon 5> coupon 2> coupon 1, coupon 3> coupon 4 (Fig. 13). We also measured the nickel concentration of the extract by atomic absorptirai spectrophotometry. The order of the nickel concentration was coupon 4> coupon 1, coupon 3> coupon 2> coupon 5 (Table 4), which was... 

For the education of the OSHA-sawy chemist, the primary purpose for assessment will usually be instruction. The teacher will be able to set assessment tasks and evaluate their results on the local level. However, there certainly will be times when it is useful to take some external measure of student success with learning the many aspects of the safety culture. External measures may take the form of a paper-and-pencil examination, such as the one described later in this chapter that has been produced by ACS DivCHED Examinations Institute. The external measure may also take the form of a performance evaluation administered by a supervisor, and such evaluations may be used in either academia or industry to satisfy a particular gate or barrier to professional advancement. 

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