A haptotaxis assay for leukocytes based on surface-bound chemokine gradients.

Abstract

The migration of leukocytes in response to chemokine gradients is an important process in the homeostasis of the human immune system and inflammation. In vivo the migration takes place on the surface of the endothelium to which the chemokine gradient is immobilized via interaction with glycosaminoglycans. To study leukocyte migration in response to surface-bound chemokines, we generated chemokine gradients by a simple stamping method: agarose stamps were soaked with chemokine solution to form continuous chemokine gradients by diffusion. These gradients could be easily transferred to a petri dish surface by stamping. We show that neutrophil granulocytes recognize these gradients and migrate toward increasing chemokine concentrations dependent on the slope of the gradient. Single-cell responses were recorded, and statistical analyses of cell behavior and migration were performed. For analysis of chemotaxis/haptotaxis, we propose a chemotactic precision index that is broadly applicable, valid, and allows for a straightforward and rapid quantification of the precision by which cells follow the direction of a given gradient. The presented technique is very simple, cost-efficient, and can be broadly applied for generating defined and reproducible immobilized gradients of almost any protein on surfaces, and it is a valuable tool to study haptotaxis.