Image-based analysis of primary human neutrophil chemotaxis in an automated direct-viewing assay

Multi-well assays based on the Boyden chamber have enabled highly parallel studies of chemotaxis—the directional migration of cells in response to molecular gradients—while direct-viewing approaches have allowed more detailed questions to be asked at low throughput. Boyden-based plates provide a count of cells that pass through a membrane, but no information about cell appearance. In contrast, direct-viewing devices enable the observation of cells during chemotaxis, which allows measurement of many parameters including area, shape, and location. Here we show automated chemotaxis and cell morphology assays in a 96-unit direct-viewing plate. Using only 12 000 primary human neutrophils per datum, we measured dose-dependent stimulation and inhibition of chemotaxis and quantified the effects of inhibitors on cell area and elongation. With 60 parallel conditions we demonstrated 5-fold increase in throughput compared to previously reported direct-viewing approaches.