Engineered Retroviruses as Fluorescent Biological Reference Particles for Small Particle Flow Cytometry

Abstract

There has been renewed interest in the use of flow cytometry for single particle phenotypic analysis of particles in the nanometer size-range such as viruses, organelles, bacteria and extracellular vesicles (EVs). However, many of these particles are smaller than 200 nm in diameter, which places them at the limit of detection for many commercial flow cytometers. The use of reference particles of diameter, fluorescence, and light-scattering properties akin to those of the small biological particles being studied is therefore imperative for accurate and reproducible data acquisition and reporting across different instruments and analytical technologies. We show here that an engineered murine leukemia virus (MLV) can act as a fluorescence reference particle for other small particles such as retroviruses and EVs. More specifically, we show that engineered MLV is a highly monodisperse enveloped particle that can act as a surrogate to demonstrate the various effects of antibody labeling on the physical properties of small biological particles in a similar diameter range.