Multiwell chamber chemotaxis assays: improved experimental design and data analysis.

The chemotaxis assay using the Boyden transfilter technique has become widely used in recent years for assessing migratory responses of a wide variety of cell types. In the study reported here we examined the migratory responses of mouse peritoneal macrophages using a multiwell chamber. The experiments were designed to analyze the components of variance in the assay method, to optimize the experimental design, and to develop objective statistical criteria for choosing among experiments with disparate results. Cell counts were obtained with the aid of an image analyzer coupled to a light microscope. Microcomputer software was developed to drive the image analyzer, collect data and conduct statistical analyses. Nested analysis of variance (ANOVA, either 2- or 3-level) was employed to partition the components of variance and F-tests were used to determine their significance. Significant sources of experimental error were identified both within and among wells and were attributed mostly to variability in the chamber/filter assembly and counting procedure. Statistical analyses demonstrated that there was significant variation among assays conducted in different multiwell chambers on the same day, among assays where the same agent was tested on different days in the same chamber, and among replicate counts of the same assay. The following recommendations were made: use ANOVA to distinguish differences due to biological effects from those due to experimental error, design experiments so that all relevant comparisons are included in the same chamber and the same assay, avoid pooling data from different assays unless ANOVA treatment variances are comparable, and when replicate assays yield disparate results choose the assay with the lowest percentage of variation due to experimental error.