Nanobodies for NETosis Tracking and Visualization

Current Protocols, vol. 5, pp. e70213

Abstract

Neutrophil extracellular traps (NETs) are pivotal in the immune response, trapping and neutralizing pathogens. The process of NET formation, or NETosis, is a critical innate immune response mechanism implicated in diverse physiological and pathological processes, including infections, chronic inflammation, and cancer. Accurate imaging of NETosis is essential for unraveling its mechanisms and therapeutic implications, although limitations of specificity and flexibility remain. In this article, we present modified nanobodies targeting Ly6C/G and histones for imaging of NETosis. Single-domain antibodies (VHHs), also known as nanobodies, targeting Ly6C/G specifically bind to neutrophils, and anti-histone nanobodies selectively target NET-associated histones, enabling NETosis monitoring. These nanobodies are engineered to optimize fluorescent probe labeling and can enable high-resolution visualization of NETosis through whole-body fluorescence tomography and/or intravital microscopy. Our approach offers an alternative for expanding the existing NETosis imaging toolkit. © 2025 Wiley Periodicals LLC. Basic Protocol 1: Cloning, cytoplasmic expression, and purification of proteins Basic Protocol 2: Fluorophore binding assay Support Protocol 1: Analysis by gel staining and immunoblotting Support Protocol 2: In-gel tryptic digestion and sample preparation for LC-MS/MS analysis Support Protocol 3: SpyCatcher003/SpyTag003 binding assay.

Authors 4

1. Nicoleta Gutu

   King Abdullah University of Science and Technology

   Affiliation as printed

   KAUST Catalysis Center (KCC), Division of Physical Sciences & Engineering King Abdullah University of Science and Technology, KAUST Thuwal Kingdom of Saudi Arabia

   These authors contributed equally to this work

2. Yuli Peng

   King Abdullah University of Science and Technology

   Affiliation as printed

   KAUST Catalysis Center (KCC), Division of Physical Sciences & Engineering King Abdullah University of Science and Technology, KAUST Thuwal Kingdom of Saudi Arabia

   These authors contributed equally to this work

3. Dominik Renn corresponding

   King Abdullah University of Science and Technology

   Affiliation as printed

   KAUST Catalysis Center (KCC), Division of Physical Sciences & Engineering King Abdullah University of Science and Technology, KAUST Thuwal Kingdom of Saudi Arabia

4. Magnus Rueping corresponding Aachen Institute for Experimental Molecular Imaging University Clinic

   King Abdullah University of Science and Technology · RWTH Aachen University

   Affiliation as printed

   Institute for Experimental Molecular Imaging University Clinic, RWTH Aachen University Aachen Germany

   KAUST Catalysis Center (KCC), Division of Physical Sciences & Engineering King Abdullah University of Science and Technology, KAUST Thuwal Kingdom of Saudi Arabia