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.

Bone-derived macrophage chemotactic factors: methods of extraction and further characterization.

Mononuclear phagocytes have been implicated as important cellular elements in the process of bone resorption. We have postulated that the recruitment and migration of mononuclear phagocytes to bone occurs via a mechanism(s) in which bone-derived chemotactic factors (BDCF) are released from foci undergoing resorption. In the experiments presented here we have used newborn mouse calvaria and examined a variety of extraction protocols, both dissociative and non-dissociative, as means of obtaining stable and reproducible chemotactic activity for mouse peritoneal macrophages. Chemotaxis and chemokinesis were assessed using a multi-well chamber modification of the Boyden transfilter method. Further, we have attempted to purify the BDCF by both molecular sieve and anion exchange chromatography. Our results indicated that non-dissociative extraction with 0.5 M EDTA in the presence of 1% DMSO yielded the most potent and reproducible chemotactic activity. The results of molecular sieve and anion exchange chromatography suggested that there were several BDCF activities in these preparations and that their molecular weights were probably in the range of from 14,000-67,000 daltons. Anion exchange chromatography also demonstrated the presence of a fraction, eluted with 2 M NaCl, with high chemotactic activity and minimal protein concentration. These observations confirmed the suggestion that there are several macrophage chemotactic factors in bone which have as yet to be identified, and suggest methods for pursuing their isolation.