Neutrophil motion in numbers: How to analyse complex migration patterns

In vivo imaging has revolutionised the study of leukocyte trafficking and revealed many insights on the dynamic behaviour of immune cells in their native environment. Neutrophil migration represents a prominent example whereby live imaging led to discovery of unanticipated cell migration patterns. These cells are the first to enter inflammatory sites and their recruitment had once been thought to be driven primarily by extrinsic signals and resolved by apoptosis in these lesions. However, in vivo imaging in zebrafish and mice indicated that neutrophils are also able to self-organise their migration to a large extent, through collective generation of gradients, in a process referred to as 'swarming', and that they can leave sites of inflammation, in a process referred to as 'reverse migration'. An important step in understanding these newly defined behaviours is the ability to detect and quantify them through statistical analysis. Here we provide a summary of considerations and recommendations for quantitative analysis of neutrophil swarming and reverse migration, with the purpose of introducing relevant analysis tools to new researchers in the field and establishing common frameworks and standards.