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.

A Haptotaxis Assay for Neutrophils using Optical Patterning and a High-content Approach

“…Rink et al . 32 recently suggested characterising chemotaxis by calculating the Chemotactic Precision Index (CPI) instead of the more common Forward Migration Index (FMI, also known simply as the Chemotactic Index or as the McCutcheon Index) 38 – 40 . They claim that CPI is a better indicator of chemotaxis because it reports simultaneously on three aspects of the cell movement: it increases as the cell moves directly toward an end point (directness), it increases if the movement is in the gradient direction (cos 2 ϕ , where ϕ is the angle between FMI and directness), and it decreases if the displacement diverts in other direction (1-|SMI|, where SMI stands for Side Migration Index).…”

“…For instance, Rink et al . 32 used hydrogel-based microcontact printing to transfer CXCL8 gradients onto a petri dish surface to study neutrophil haptotaxis. While agarose stamping of a diffusion gradient is simple to perform, it results in only a short-lived protein gradient induced by the deposition of thin agarose layers on a substrate.…”

Convergent and Divergent Migratory Patterns of Human Neutrophils inside Microfluidic Mazes

“…Although microfluidic devices have been developed specifically to dissect the differential contributions of superimposed soluble and haptotactic gradients to cell migration 37 , this was not possible using our current maze device design. Surface-bound IL-8 has been shown to induce random migration by human neutrophils 38 , 39 . A simpler device was therefore employed to compare the motility induced by soluble and bound gradients.…”

Deconstructing Immune Microenvironments of Lymphoid Tissues for Reverse Engineering

“…Secretion and immobilization of soluble IL‐8 on localized GAG‐presenting BECs and nearby connective tissue causes neutrophils to preferentially crawl and invade sites of inflammation. Rink et al, experimentally showed neutrophil CXCR1/CXCR2‐mediated haptotaxis using custom agarose stamps to micropattern IL‐8 binding sites on glass substrates . Using a laser‐assisted protein adsorption method, to reproduce substrate bound formyl‐peptide concentration gradients, formyl peptide receptor (FPR)‐mediated haptotaxis was demonstrated .…”