Temporal assay

Dearman, R.J. et al., Temporal stability of local lymph node assay responses to hexyl cinnamic aldehyde, J. Appl. Toxicol., 18, 281, 1998. 

The protocol outlined in this chapter has been optimized for processing a large number of mouse embryos simultaneously. Since a lot of protocols include optional or even unnecessary steps largely for historical reasons, this protocol intends to maximize the efficiency of assays and minimize the temporal and financial burden, which are often the serious issues in a large-scale experiment. For reference, the alternative procedures, which were omitted or simplified in this protocol, are described in Subheading 4 as potential troubleshooting tips. 

Synaptic neurotransmission in brain occurs mostly by exocytic release of vesicles filled with chemical substances (neurotransmitters) at presynaptic terminals. Thus, neurotransmitter release can be detected and studied by measuring efflux of neurotransmitters from synapses by biochemical methods. Various methods have been successfully employed to achieve that, including direct measurements of glutamate release by high-performance liquid chromatography of fluorescent derivatives or by enzyme-based continuous fluorescence assay, measurements of radioactive efflux from nerve terminals preloaded with radioactive neurotransmitters, or detection of neuropeptides by RIA or ELISA. Biochemical detection, however, lacks the sensitivity and temporal resolution afforded by electrophysiological and electrochemical approaches. As a result, it is not possible to measure individual synaptic events and apply quantal analysis to verify the vesicular nature of neurotransmitter release. 

A medium throughput approach to evaluating sodium channel activity is the measurement of sodium flux across cell membranes [103]. In these experiments, a tracer that permeates the channel and is easily quantifiable is used to analyze sodium influx. Traditionally, radioactive tracers such as 22Na+ or [14C]guanidinium have been used. Alternatively, Li+ can be used as a tracer and analyzed by atomic absorption spectrometry. Sodium flux assays can be used to test approximately 105 compounds per year. They offer a robust readout of channel activity, but lack voltage control and temporal resolution. To examine sodium channel blockade by measuring sodium flux,... 

Then, given a model for data from a specific drug in a sample from a population, mixed-effect modeling produces estimates for the complete statistical distribution of the pharmacokinetic-dynamic parameters in the population. Especially, the variance in the pharmacokinetic-dynamic parameter distributions is a measure of the extent of inherent interindividual variability for the particular drug in that population (adults, neonates, etc.). The distribution of residual errors in the observations, with respect to the mean pharmacokinetic or pharmacodynamic model, reflects measurement or assay error, model misspecification, and, more rarely, temporal dependence of the parameters. 

Conventional free interface diffusion achieves high transient levels of supersaturation, but has a complicated spatial/temporal gradient due to the constant cross-section of the capillary. This gradient couples the kinetics and thermodynamics of traditional free interface diffusion assays in a way that pFID does not. 

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