Cultures experimental design

In the selection of an appropriate cell culture system, a number of criteria must be considered (Table 3). These include not only the characteristics of the cell type but also the controllable parameters of the complete transport system such as the permeants, the filter properties, and the assay conditions. In general, most transport experiments employ the experimental design shown schematically in Figure 4 with modifications as discussed below. Typically, the desired cell is seeded onto some sort of semipermeable filter support and allowed to reach confluence. The filter containing the cell monolayer separates the donor and receiver... 

The use of appropriate experimental design can provide definitive evidence that P-gp-mediated efflux is altering the transport of a compound and can provide further mechanistic information regarding the transport of a compound. Recently it has been appreciated that P-gp efflux can be a potential source for chug interactions and in vitro experimentation can be very helpful to understand potential liability. The techniques described in this section can be used with any tissue culture transport model. 

The volume of culture medium used per well should be adequate to nourish the cells for the entire duration of the experiment. The cost of culture medium is usually not a driving factor for experimental design. If the experimental design uses long incubation periods, larger volumes may be required to provide adequate nutrients and avoid any problems due to evaporation. 

Plant materials Burley tobaccos (Nicotiana tabacum L. cv KY 14 and cv KY 17) were grown at various times in the soil floor of a greenhouse. Recommended cultural and fertilization practices were followed (11). A randomized complete block experimental design... 

In contrast, in tighter epithelia such as Caco-2 and in artificial membranes such as HDM, the permeability of the ionized forms of the drugs and the paracellular permeability are lower or insignificant, respectively. These findings will have implications in the experimental design and data interpretation of pH-dependent drug transport experiments in cell culture models as well as in artificial membrane models, such as HDM and PAMPA. 

Biological assays are often noisy and laborious. With careful application of experimental design, cell culture bioassays can be made quite accurate and precise. The core information needed for validation can come from two experiments. One experiment studies accuracy and precision followed by a variance component analysis and a summary table that describes the expected performance of the system at various levels of replication. A second experiment uses a minimal fractional factorial design to study robustness, followed by a comparison of confidence intervals on effect sizes with a previously established indifference zone. 

Finally, the QCM can not only be used in a sensory mode but also as an actuator. It has been recently shown by Dultsev and coworkers [57] that virus particles deposited on the resonator surface may be displaced by increasing the shear amplitude of the resonator. Thus, it seems plausible that the resistance of cell-substrate interactions to lateral shear forces may be inferred from QCM measurements when the shear amplitude is increased to invasive magnitudes. The ease of the measurement, which can be automated and multiplexed, the rather simple experimental design, as well as the unique experimental access to the interface between living cells and technical substrates is very likely to create growing interest within the cell culture community for these new experimental options. 

Characteristic of immune function evaluations, there is considerable diversity in the approaches to proliferation assays, from the experimental design to the analytical method. Experimental procedures necessitate sterile technique and cell culture expertise to ensure accurate assessment of the cellular response to a particular stimulant. Culture conditions vary depending on the cell source and type of stimulant, but generally are conducted at 5% CO2,37 °C for 48 to 96 hours. The source of lymphocytes may be from peripheral blood, spleen. 

Prior to each experiment, foraminifers were incubated in calcein (Bernhard et al. 2004). Calcein staining prior to culture chamber inoculation provided a way to distinguish pre-experimental individuals (and pre-experimental chambers within an individual) from individuals or chambers that calcified under controlled conditions during the experiments. At the start of each culturing experiment 80-100 calcein-stained specimens (multiple species from a single site, > 90 p,m in diameter) were added to cell tissue culture cups (8 mL volume, 8 p,m nonunal pore diameter) housed in acrylic culture chambers. There were nine culture chambers in the 2001 experiment and twelve in the 2002 experiment. Each culture cup contained a l-2mm-thick layer of silt-sized silicon dioxide to provide the physical substrate required for foraminiferal growth the culture chamber design and use of an artificial substrate minimize formation of sedimentary microhabitats within the culture cups (Wilson-Finelli et al. 1998 Havach et al. 2001 Hintz et al. 2004). 

A new Experimental Design Chart must be used for this Indirect Immunocytochemistry Block-Between (Table 12.1), because it differs from the chart used previously. Here, the experiment uses cultured cells on coverslips and two 1° antibodies made in mouse, mouse anti-Ag A and mouse anti-Ag B. A new row on the Chart is needed, 4. Block-between, to list two new reagents used to block-between the two 1° antibodies. Normal mouse serum is used at 1 20, which is sufficient to block in most cases. The anti-mouse Fab molecule is used at 20 p.g/ml. Incubations for each of these steps is for 1 h with six rinses after each incubation. Note these blocking incubations must be performed in the order of normal mouse serum first and anti-mouse Fab second. 

Following this experimental design, approximately 700 plant materials have been evaluated and this has resulted in over 6,500 bioassay results. A number of active principles have been obtained that are active with the in vitro test systems, and several of the isolates have retained activity by preventing formation of preneoplastic lesions in mammary organ culture. Thus far, three lead compounds have been shown to mediate considerable cancer chemopreventive activity in full-term tumorigenesis models (28,34,45,46) and are being studied in more advanced test systems. We remain hopeful that one or more discoveries resulting from this project will be deemed worthy of human intervention trials. 

Experimental design model monoxenic culture in a two-compartment system... 

If the experimental design includes co-infiltration of two or three constructs in one plant, combine the acetosyringone-treated cultures at the end of the 3 h incubation in acetosyringone. The ratio for mixing the cultures for coinfiltration is 1 1... 

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