Setting Up Controls
Controls and Other Sites in Cell-Activity Assays
We assume that a cell line and chemical chemotactic factor have been selected as the focus of the assay, and that the effects of the chemical on the cells’ activity will be demonstrated using an instrument that includes sites at which the chemical is in contact with the under side of a membrane filter while a suspension containing the cells lies on top of the filter. For simplicity, we will also assume that chemotaxis (directional cell motion along a concentration gradient of a chemical) is the cell activity being detected and measured. (Neuro Probe instruments have many applications other than straightforward chemotaxis assays—for example experiments focusing on transmigration through an endothelial layer or metastasis through extracellular matrix proteins. Much of the following discussion can be adapted to apply to such applications.) There is a general discussion of chemotaxis experiment design at cell-activity assays.
A cell-activity assay must have
unknown sites and control sites. Control sites must include
negative control sites, normally will include
positive control sites, and in many cases will include
chemokinetic control sites. Often a set or series of related sites is used, for example a
dose-response series or a
calibration series. Each kind of site and series is discussed in turn.
Unknown sites have cell suspension above the filter and a solution containing the unknown chemotactic factor (the chemical whose properties are being tested in the assay) below it. The purpose of the assay is to measure the effects of the chemotactic factor on the cells by measuring the extent to which the number of cells migrating through the membrane filter is influenced by the presence of the chemotactic factor on the under side.
Negative control sites have cell suspension above the filter and cell-suspension media, but no chemotactic factor, below, so the cells are not stimulated by a chemotactic factor. At every site, random migration of unstimulated cells will account for some of the cells that pass through the filter. Migrated cells at negative control sites show the extent of unstimulated random migration, which can then be differentiated from migration induced or influenced by the chemotactic factor. An assay is designed to maximize the differential between the number of migrated cells at unknown and negative control sites.
Positive control sites have cell suspension above the filter and below it a solution containing a known chemoattractant (a chemotactic factor whose influence on the cells being studied is already known). Including positive control sites in an assay allows the effects of the unknown chemotactic factor to be compared with those of a known chemoattractant.
Chemokinetic control sites have a solution containing the unknown chemotactic factor, at the same concentration, not only below the filter but above it as well—in the suspension with the cells. At these sites there is no concentration gradient of chemotactic factor in the pores—between the top and bottom surfaces of the filter. Chemotactic factors cause chemokinesis (increased activity in cells stimulated by a chemical), and chemokinesis by itself will increase the likelihood of cells moving through the filter. Migrated cells at the chemokinetic control sites show the extent of non-chemotactic chemokinetic migration, which can then be differentiated from chemotaxis.
A dose-response series is a series of sites that contains, below the filter, a serial dilution of a chemoattractant. Cell response in the form of chemotaxis varies not only with the chemoattractant, but also with the nature of the concentration gradient in filter pores and around the tops of the pores. If the solution below the filter is too weak in chemoattractant, the concentration gradient may not stimulate the cells; if the concentration below the filter is too strong, chemoattractant around the tops of the pores may be so concentrated that the stimulus is not directional. A dose-response series is used to determine what concentration of a chemoattractant will result in optimum chemotactic response on the part of the cells being tested.
A checkerboard assay combines elements of chemokinetic controls and dose response. The chemokine is added to both the upper and lower wells in alternating concentrations, i.e., the concentration in the upper well decreases as the concentration in the lower well increases. Thus the chemoattracant and chemokinetic effects may be differentiated.
A calibration series is a series of sites that contains, below the filters, a serial dilution of cell suspension. No solution is present above the filters. Microplate based automated detection systems do not count individual cells; they correlate the number of cells in a population with the level of some other property of that population, as measured by a detection device (optical density in densitometric readers, for example, or degree of fluorescence in fluorescence readers). A calibration series provides a set of sites with a range of known cell numbers in microplate wells below the filter. It is used to determine the relationship between the number of cells at each site and the level registered by the detection device. This provides a series of correlations that can be used as a standard or metric in determining the number of cells at other assay sites.