EdU tracking of leukocyte recruitment in mouse models of ischemic stroke and sterile lung inflammation.

Abstract

The discovery of copper(I)-catalyzed azide-alkyne cycloaddition (click chemistry) has significantly advanced the detection of proliferating cells by utilizing 5-ethynyl-2'-deoxyuridine (EdU). EdU, a thymidine analogue, is incorporated into DNA during replication and detected by the direct reaction with an azide-conjugated fluorophore. Traditionally, dividing cells are labeled using 5-bromodeoxyuridine (BrdU), another nucleotide analogue. However, BrdU detection is a harsh method that requires substantial sample processing, unlike EdU detection. EdU is classically used to identify proliferating cells; however, we report a streamlined methodology that uses EdU to label and track leukocyte recruitment that is compatible with flow cytometry and microscopy and preserves transgenic fluorophores. EdU labeling was performed in two different models of sterile inflammation: ischemic stroke and hydrochloric acid aspiration. EdU injection was timed to differentially label circulating monocytes, neutrophils and T cells. Tissue analysis showed EdU-positive monocytes and T cells were enriched in both inflammatory models. This suggests that recently divided monocytes and T cells are preferentially recruited to these vascular beds during inflammation and highlights the utility of this labeling approach to track leukocyte subtypes longitudinally during inflammation.