Abstract
The defining biology that distinguishes neutrophil extracellular traps (NETs) from other forms of cell death is unresolved, and techniques which unambiguously identify NETs remain elusive. Raman scattering measurement provides a holistic overview of cell molecular composition based on characteristic bond vibrations in components such as lipids and proteins. We collected Raman spectra from NETs and freeze/thaw necrotic cells using a custom built high-throughput platform which is able to rapidly measure spectra from single cells. Principal component analysis of Raman spectra from NETs clearly distinguished them from necrotic cells despite their similar morphology, demonstrating their fundamental molecular differences. In contrast, classical techniques used for NET analysis, immunofluorescence microscopy, extracellular DNA, and ELISA, could not differentiate these cells. Additionally, machine learning analysis of Raman spectra indicated subtle differences in lipopolysaccharide (LPS)-induced as opposed to phorbol myristate acetate (PMA)-induced NETs, demonstrating the molecular composition of NETs varies depending on the stimulant used. This study demonstrates the benefits of Raman microscopy in discriminating NETs from other types of cell death and by their pathway of induction.