Population-based assays

Filter assays have been very popular for identifying soluble compounds that stimulate locomotion (reviewed in [424, 426]). Two chambers are separated by a filter with pore sizes less than the diameter of the cells, such that cells must actively migrate to get through. The apparatus is oriented such that cells are put in the top chamber and can settle onto the filter. Solution with chemoattractant is put in the bottom chamber, and as it diffuses through the pores a concentration gradient is established such that the cells crawl through the filter. There are two main types of filter 

Cel invasion assays have been developed for monitoring migration in three dimensions through physiologically relevant matrices [47, 148, 

To the typical cellular or molecular biologist and physiologist this equation may look quite formidable. However the terms have physical meanings that can be readily understood. The equation describes the cell number density as a function of spatial position, x, and time, t, such that dc/dt represents the rate at which cell number density at some position X changes with time. The first term on the right side of the equation quantifies the change in cell number density due to random migration. 

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Optophoresis has the advantage over other cell-based assays in that a small population of cells is used, and individual data on the drug response of each cell in the population is acquired. In this way subpopulations in a heterogeneous sample can be detected and analyzed. In addition, because of the low number of cells required, the cells do not need to be grown out or manipulated in any way. They are merely incubated with the drug ex vivo for the appropriate time to obtain a cellular response and then measured. 

Anderson DJ, Puttfarcken PS, Jacobs 1, Faltynek C (2000) Assessment of nicotinic acetylcholine receptor-mediated release of [ H]-norepinephrine from rat brain slices using a new 96-well format assay. Neuropharmacology 39 2663-2672 Anney RJ, Olsson CA, Lotfi-Miri M, Patton GC, Williamson R (2004) Nicotine dependence in a prospective population-based study of adolescents the protective role of a functional tyrosine hydroxylase polymorphism. Pharmacogenetics 14 73-81 Auerbach A, Akk G (1998) Desensitization of mouse nicotinic acetylcholine receptor channels. 

Analogous to cell viability measurements, many of the same basic concepts apply to the development of cell-based assays for apoptosis. The length of incubation of cells with the test compound is among the most important issues to address and optimize. The length of incubation is important because the markers of apoptosis may be present for relatively brief transient periods and subsequently disappear as the population of cells undergoes secondary necrosis. The induction of measurable caspase activity can occur in only a few minutes or can take days, depending on the model cell line, type of inducer, and effective concentration inside the cells. 

Cytometry assays treat each cell as a single entity and score for population changes of cells with certain phenotypic characteristics. Fluorescence-activated cell sorting (FACS) is the most common format for cytometry-based assays. FACS requires large numbers (>105) of cells and does... 

The resistance of a surface to the irreversible non-specific adsorption of proteins is an essential requirement for its application as a substrate in a high-throughput device for carrying out diagnostic assays, in which cell-based assays for screening libraries of drug candidates are performed in a chip format. The protein resistance of the surface allows a cell population... 

Predictive in vitro test methods are as well developed as nonanimal test alternatives, including specific cell-based assays.22 Such cellular assays include the culture of keratinocytes that represent very often the first cells in the skin to encounter potential reactive chemicals. Purified keratinocytes are cultured with a specific test chemical, and the production of proinflammatory cytokines or chemokines are then measured. Another cell population that can be used to test for chemicals inducing contact allergy are Langerhans cells (LCs). LCs are the... 

SNPs were picked for association studies on the basis of allele frequencies and results from in silico predictions, and association studies were carried out in various populations, including association of PXR variants with PXR and CYP3A4 expression in human livers and primary human hepatocytes. In addition, the PXR variants were cloned, expressed, and their functional consequence determined in cell-based assays. 

In conventional population cell-based assays, sample preparation involves several steps, including lysis, filtration of non-solubUized cellular material, labeling of the analytes of interest, and sample purification. However, to prevent dilution of the extremely small samples in single-cell analysis, filtration and purification are not typically integrated in chemical cytometiy methods. This section briefly describes the various cell lysis techniques which have been implemented in microfluidics for more detail, please see On-Chip Cell Lysis. 

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