# Rapid autofluorescence flow cytometric analysis of agonist-induced neutrophil and eosinophil polarization reveals novel insights into 5-oxo-ETE-mediated granulocyte activation

## TL;DR

This paper introduces a new method using flow cytometry to study how neutrophils and eosinophils change shape in response to specific signals, without using external probes that might alter cell behavior.

## Contribution

The study introduces a novel antibody-free flow cytometry approach using autofluorescence and forward scatter to rapidly assess granulocyte polarization and activation.

## Key findings

- Stimulation with fMLF caused neutrophil polarization, marked by FSC shifts, reduced CD62L, increased CD11b, and ROS production.
- Autofluorescence-based gating enabled identification of eosinophils in mixed granulocyte populations and revealed distinct activation patterns.
- 5-oxo-ETE activated both neutrophils and eosinophils, while eotaxin selectively activated eosinophils.

## Abstract

Minimizing unintended granulocyte activation while measuring functional responsiveness is essential, as the use of external probes, antibodies, or fluorescent dyes can potentially alter cellular responsiveness. To address this, we employed an antibody-free flow cytometry approach that measures forward scatter (FSC) to detect variations in cell-size, morphology, and shape; some key indicators of neutrophil and eosinophil activation. Human peripheral blood neutrophils, containing contaminating eosinophils, were isolated using discontinuous Percoll gradients and pre-treated with receptor antagonists [e.g., cyclosporin-H (an FPR1 antagonist) and CP105696 (a BLT1 receptor antagonist)] prior to stimulation with agonists such as fMLF (an FPR1 agonist) and LTB4 (a BLT1 agonist). Furthermore, fMLF stimulation resulted in a loss of CD62L and an increase in CD11b expression along with an increase in intracellular ROS production compared to control, as analysed using flow cytometry. Imaging flow cytometry, together with FSC analysis, enabled assessment of cell polarization and associated morphological changes. Importantly, autofluorescence-based gating allowed for the identification of contaminating eosinophils within the mixed granulocyte population, allowing parallel assessment of shape-change in both neutrophils and eosinophils in response to the same ligands. Stimulation of neutrophils with fMLF resulted in distinct FSC shifts compared to unstimulated controls across all flow cytometers tested, which were inhibited by cyclosporin-H, but not CP105696. Morphological analysis confirmed these changes corresponded with increased cell area and perimeter and decreased circularity, hallmarks of cell polarisation. Additionally, selective activation of eosinophils (but not neutrophils) by eotaxin, and dual activation of both cell types by the arachidonic acid metabolite 5-oxo-ETE, were confirmed through specific gating strategies. Taken together, these findings support the use of FSC-based flow cytometry as a rapid, scalable and effective method for evaluating granulocyte polarisation and screening candidate therapeutics targeting immune cell activation in disease contexts.

The online version contains supplementary material available at 10.1186/s12950-025-00472-8.