RT Journal Article SR Electronic T1 Engineered Retroviruses as Fluorescent Biological Reference Particles for Nanoscale Flow Cytometry JF bioRxiv FD Cold Spring Harbor Laboratory SP 614461 DO 10.1101/614461 A1 Vera A. Tang A1 Anna K. Fritzsche A1 Tyler M. Renner A1 Dylan Burger A1 Joanne A. Lannigan A1 George C. Brittain A1 Christian V. Ouellet A1 Edwin van der Pol A1 Marc-André Langlois YR 2019 UL http://biorxiv.org/content/early/2019/04/19/614461.abstract AB Nanoscale flow cytometry (NFC) is becoming a method of choice for the phenotypic analysis of viruses 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 size, fluorescence, and light-scattering properties similar to that of the small particles of interest 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 which robustly satisfies these criteria. MLV can be engineered to express proteins of interest at high, but biologically relevant levels, on the surface of its viral envelope, which is derived from the cell plasma membrane. These recombinant proteins display consitent expression in the released virus population, and are readily labeled by antibody, making engineered MLVs effective and customizable positive controls for antibody-labeling assays. This feature also enables the use of fluorescence to quantify surface protein molecule expression, as well as compare different antibody fluorophore conjugates and concentrations to determine optimal resolution of a small particle populations. In this study, we also reveal that fluorophore labeling can increase the size and skew the refractive index of small particles, thereby reinforcing the necessity for using suitable reference particles for NFC analyses. Our study showcases that MLV is a monodisperse EV surrogate that can be used to control and evaluate the various effects of antibody labelling on the physical properties of small vesicular particles.